BIOCHEMISTRY OF HORMONES. CLASSIFICATION DUE TO THEIR CHEMICAL NATURE AND PHYSIOLOGICAL ACTION, MECHANISM OF INFLUENCE AT TARGET CELLS. BIOCHEMISTRY OF THYROID AND PARATHYROID GLANDS HORMONES. BIOCHEMICAL CHANGES IN THE ORGANISM IN ENDOCRINE VIOLATIONS. INTRODUCTION OF METABOLISM. GENERAL PATHWAYS OF METABOLISM IN THE ORGANISM. BIOENERGETICS. KREBS CYCLE, BIOLOGICAL OXIDATION, OXIDATIVE PHOSPHORYLATION
The survival of multicellular organisms depends on their ability to adapt to a constantly changing environment. Intercellular communication mechanisms are necessary requirements for this adaptation. The nervous system and the endocrine system provide this intercellular, organism- wide communication. The nervous system was originally viewed as providing a fixed communication system, whereas the endocrine system supplied hormones, which are mobile messages. In fact, there is a remarkable convergence of these regulatory systems. For example, neural regulation of the endocrine system is important in the production and secretion of some hormones; many neurotransmitters resemble hormones in their synthesis, transport, and mechanism of action; and many hormones are synthesized in the nervous system.
The word “hormone” is derived from a Greek term that means to arouse to activity. As classically defined, a hormone is a substance that is synthesized in one organ and transported by the circulatory system to act on another tissue.
Hormones are chemical signaling substances. They are synthesized in specialized cells that are often associated to form endocrine glands. Hormones are released into the blood and transported with the blood to their effector organs.
In the organs, the hormones carry out physiological and biochemical regulatory functions. In contrast to endocrine hormones, tissue hormones
are only active in the immediate vicinity of the cells that secrete them. The distinctions between hormones and other signaling substances (mediators, neurotransmitters, and growth factors) are fluid. Mediators is the term used for signaling substances that do not derive from special hormone- forming cells, but are formbymany cell types. They have hormone-like effects in their immediate surroundings. Histamine and prostaglandins are important examples of these substances.
Neurohormones and neurotransmitters are signaling substances that are produced and released by nerve cells.
Endocrine glands: hypothalamus, pituitary, epiphysis, thyroid, parathyroid, thymus, pancreas, adrenal glands, sex glands
Hormonoids (tissue hormones, histohormones) - organic trace substances produced by different cells of different tissues (not by specific glands) that regulate metabolism on the local level (some hormonoids are produced in the blood too (serotonin, acetylcholine).
2. Functions of hormones.
Hormones regulate the following processes:
Growth and differentiation of cells, tissues, and organs These processes include cell proliferation, embryonic development, and sexual differentiation— i. e., processes that require a prolonged time period and involve proteins de novo synthesis. For this reason, mainly steroid hormones which function via transcription regulation are active in this field
Metabolic pathways Metabolic regulation requires rapidly acting mechanisms. Many of the hormones involved therefore regulate interconversion of enzymes. Themain processes subject to hormonal regulation are the uptake and degradation of storage substances (glycogen, fat), metabolic pathways for biosynthesis and degradation of central metabolites (glucose, fatty acids, etc.), and the supply of metabolic energy.
Digestive processes Digestive processes are usually regulated by locally acting peptides (paracrine), but mediators, biogenic amines, and neuropeptides are also involved.
Maintenance of ion concentrations (homeostasis) Concentrations of Na+, K+, and Cl– in body fluids, and the physiological variables dependent on these (e. g. blood pressure), are subject to strict regulation. The principal site of action of the hormones involved is the kidneys, where hormones increase or reduce the resorption of ions and recovery of water. The concentrations of Ca2+ and phosphate, which form the mineral substance of bone and teeth, are also precisely regulated. Many hormones influence the above processes only indirectly by regulating the synthesis and release of other hormones (hormonal hierarchy).
3. Endocrine, paracrine, and autocrine hormone effects.
Hormones transfer signals by migrating from heir site of synthesis to their site of action. They are usually transported in the blood. In this case, they are said to have an endocrine effect (example: insulin). By contrast, tissue hormones, the target cells for which are in the immediate vicinity of the glandular cells that produce them, are said to have a paracrine effect (example: gastrointestinal tract hormones). When signal substances also pass effects back to the cells that synthesize them, they are said to have an autocrine effect (example: prostaglandins). Autocrine effects are often found in tumor cells, which stimulate their own proliferation in this way. Insulin,which is formed in the B cells of the pancreas, has both endocrine and paracrine effects. As a hormone with endocrine effects, it regulates glucose and fat metabolism. Via a paracrinemechanism, it inhibits the synthesis and release of glucagon from the neighboring A cells.
4. Classification of hormones.
The animal organism contains more than 100 hormones and hormone-like substances, which can be classified either according to their structure or according to their function. In chemical terms, most hormones are:
Ø hormones of protein structure: all hormones of anterior pituitary (except ACTH), insulin, parathyroid hormone;
Ø hormones of peptide structure: ACTH, calcitonin, glucagon,hormones of posterior pituitary, factors of hypothalamus, thymozin;
Ø steroid hormones: adrenal cortical steroids, sex hormones;
Ø hormones - derivatives of amino acid: thyroid hormones, adrenal medulla hormones, epiphysis hormones;
Ø .hormones derivatives of unsaturated fatty acid: prostaglandins.
5. Mechanism of action
A. Mechanism of action of lipophilic hormones
Lipophilic signaling substances include the steroid hormones, calcitriol, the iodothyronines (T3 and T4), and retinoic acid. These hormones mainly act in the nucleus of the target cells, where they regulate gene transcription in collaboration with their receptors and with the support of additional proteins (known as coactivators and mediators). There are several effects of steroid hormones that are notmediated by transcription control. These alternative pathways for steroid effects have not yet been fully explained. In the blood, there are a number of transport proteins for lipophilic hormones. Only the free hormone is able to penetrate the membrane and enter the cell. The hormone encounters its receptor in the nucleus (and sometimes also in the cytoplasm). The receptors for lipophilic hormones are rare proteins. They occur in small numbers (103–104 molecules per cell) and show marked specificity and high affinity for the hormone (Kd = 10–8–10–10 M). After binding to the hormone, the steroid receptors are able to bind as homodimers or heterodimers to control elements in the promoters of specific genes, from where they can influence the transcription of the affected genes—i. e., they act as transcription factors. The illustration shows the particularly well-investigated mechanism of action for cortisol, which is unusual to the extent that the hormone–receptor complex already arises in the cytoplasm. The free receptor is present in the cytoplasm as a monomer in complex with the chaperone hsp90. Binding of cortisol to the complex leads to an allosteric conformational change in the receptor, which is then released from the hsp90 and becomes capable of DNA binding as a result of dimerization. In the nucleus, the hormone–receptor complex binds to nucleotide sequences known as hormone response elements (HREs). These are short palindromic DNA segments that usually promote transcription as enhancer elements. The illustration shows the HRE for glucocorticoids (GRE; “n” stands for any nucleotide). Each hormone receptor only recognizes its “own” HRE and therefore only influences the transcription of genes containing that HRE. Recognition between the receptor and HRE is based on interaction between the amino acid residues in the DNA-binding domain (B) and the relevant bases in the HRE (emphasized in color in the structure illustrated). As discussed on p. 244, the hormone receptor does not interact directly with the RNA polymerase, but rather—along with other transcription factors—with a coactivator/mediator complex that processes all of the signals and passes them on to the polymerase. In this way, hormonal effects lead within a period of minutes to hours to altered levels ofmRNAs for key proteins in cellular processes (“cellular response”).
B. Mechanism of action of hydrophilic hormones
The messages transmitted by hydrophilic signaling substances are sent to the interior of the cell by membrane receptors. These bind the hormone on the outside of the cell and trigger a new second signal on the inside by altering their conformation. In the interior of the cell, this secondary signal influences the activity of enzymes or ion channels. Via further steps, switching of the metabolism, changes in the cytoskeleton, and activation or inhibition of transcription factors can occur (“signal transduction”) can occur.
6. Examples of peptide hormones and proteohormones
Hormones of hypothalamus (releasing and inhibitory factors), structure, mechanism of action.
Hypothalamus has the wide anatomic links with other parts of the brain. Therefore in different mental disorders there is the change of secretion of hypothalamus hormones.
Two groups of hormones are produced by hypothalamus corresponding to the anterior and posterior pituitary.
Hypothalamus and posterior pituitary. 3 peptides are synthesized in the hypothalamus that pass to the posterior pituitary along axons where they are accumulated: oxytocin, vasopressin (antidiuretic hormone) and neurophysin. The later binds the oxytocin and vasopressin and promotes their transportation to the pituitary.
Hypothalamus and anterior pituitary. Hypothalamus is connected with the anterior pituitary by the net of blood capillaries, so called hypothalamic portal system. Hypothalamus produces very active peptide compounds that pass via this portal system to anterior pituitary and stimulate or oppress the secretion of tropic hormones. Compounds stimulating the secretion are called releasing factors. 7 releasing factors are known according to the amount of tropic hormones of anterior pituitary:
- corticotropin-releasing factor
- thyrotropin-releasing factors
- somatotropin-releasing factors
- follicletropin-releasing factor
- luteotropin-releasing factor
- prolactotropin-releasing factor
- melanotropin-releasing factor.
Hypothalamus also secretes substances called inhibitory factors or statins, which can inhibit release of the some pituitary hormones. 3 inhibitory factors are known today:
Releasing and inhibitory factors are produced in only minute amounts.
Hormones of pituitury, structure, mechanism of action.
Tropic hormones are produced by the anterior pituitary. Usually tropic hormones not directly regulate the metabolism but act on the peripheral endocrine glands.
Somatotropic hormone (STH, growth hormone).
Chemical structure: simple protein
The intensity of secretion is regulated by the relationship between the somatotropic-releasing factor and somatostatin.
The main function of somatotropic hormone - stimulation of growth. Hormone is necessary for the bone tissue formation, for the muscle tissue growth, for the formation of peculiarities of men and women body.
Somatotropic hormone can act both directly on the metabolism and indirectly stimulating the synthesis of somatomedines (specific protein growth factors which are synthesized in liver).
The effect of somatotropic hormone on
- protein metabolism: stimulates the passing of amino acids into the cells;
activates the synthesis of proteins, DNA, RNA.
- carbohydrate metabolism: activates the insulinase of liver;
inhibits the conversion of lipids to carbohydrates;
activates the exit of glucose from liver;
inhibits the entry of glucose into the cells.
- lipid metabolism: stimulates lipolisis;
stimulates the oxidation of fatty acids.
The deficiency of somatotropic hormone in children age causes nanism. Nanism - proportional underdevelopment of all body.
The deficiency of somatotropic hormone in adult persons hasn’t clinical symptoms.
The excess of somatotropic hormone in children age causes gigantism.
The excess of somatotropic hormone in adult persons causes acromegalia (disproportional development of the separate body parts).
Thyrotropic hormone (TTH).
Chemical structure: glicoprotein.
This hormone is necessary for the normal functions of thyroid glands.
Thyrotropic hormone promotes:
- accumulation of iodine in thyroid;
- including of iodine into the tyrosine;
- synthesis of thyroxine and triiodothyronine.
Adrenocorticotropic hormone (ACTH).
Chemical structure: polipeptide.
This hormone is necessary for the normal functions of adrenal cortex. It enhances the formation of steroid hormones and their secretion into the blood.
ACTH has also the melanocyte-stimulating activity.
Excessive secretion of ACTH causes the Icenko-Kushing disease (symptoms of hypercorticism, hyperpigmentation).
Follicle stimulating hormone (FSH).
Chemical structure: glycoprotein.
Function: stimulates the function of follicles (oogenesis) in women and spermatogenesis in men.
Luteinizing hormone (LH).
Chemical structure: glycoprotein.
Function: stimulates the formation of yellow body in women and testosterone secretion in men.
Chemical structure: protein.
Functions: - stimulates lactation;
- stimulates function of yellow body (secretion of progesterone);
- promotes formation of mother instinct;
- stimulates the formation of prostate glandular tissue in men.
Chemical structure: protein.
Functions: - stimulates the mobilization of lipids from depot;
- decreases the Ca amount in blood;
- has the melanocyte-stimulating activity.
Chemical structure: peptide.
Functions: - activates the hyaluronidase. This enzyme splits the hyaluronic acid. The permeability of membranes is increased and reabsorption of water in kidneys is increased too. As result the day diuresis is decreased;
- narrows arterioles and capillaries and increases the blood pressure.
The deficiency of vasopressin in organism causes diabetes insipidus. Clinical symptoms - poliuria, dehydration of the organism, low density of the urine.
Chemical structure: peptide.
Functions: stimulates the contraction of smooth muscles, especially the muscles of uterus and muscle fibres of alveoluses of mammas.
Oxytocin is used for delivery stimulation, for stop of bleeding after delivery, for stimulation of lactation.
Numerically the largest group of signaling substances, these arise by protein biosynthesis. The smallest peptide hormone, thyroliberin (362 Da), is a tripeptide. Proteohormones can reach masses of more than 20 kDa—e. g., thyrotropin (28 kDa). Similarities in the primary structures of many peptide hormones and proteohormones show that they are related to one another. They probably arose from common predecessors in the course of evolution. Thyroliberin (thyrotropin-releasing hormone, TRH) is one of the neurohormones of the hypothalamus. It stimulates pituitary gland cells to secrete thyrotropin (TSH). TRH consists of three amino acids, which are modified in characteristic ways.
Thyrotropin (thyroid-stimulating hormone, TSH) and the related hormones lutropin (luteinizing hormone, LH) and follitropin (follicle-stimulating hormone, FSH) originate in the adenohypophysis. They are all dimeric glycoproteins with masses of around 28 kDa. Thyrotropin stimulates the synthesis and secretion of thyroxin by the thyroid gland.
Insulin is produced and released by the B cells of the pancreas and is released when the glucose level rises. Insulin reduces the blood sugar level by promoting processes that consume glucose— e. g., glycolysis, glycogen synthesis, and conversion of glucose into fatty acids. By contrast, it inhibits gluconeogenesis and glycogen degradation.
Glucagon, a peptide of 29 amino acids, is a product of the A cells of the pancreas. It is the antagonist of insulin and, like insulin, mainly influences carbohydrate and lipid metabolism. Its effects are each opposite to those of insulin. Glucagon mainly acts via the second messenger cAMP
7. Signaling substances derived from amino acids
Histamine, serotonin, melatonin, and the catecholamines dopa, dopamine, norepinephrine, and epinephrine are known as “biogenic amines.” They are produced from amino acids by decarboxylation and usually act not only as hormones, but also as neurotransmitters. Histamine, an important mediator (local signaling substance) and neurotransmitter, is mainly stored in tissue mast cells and basophilic granulocytes in the blood. It is involved in inflammatory and allergic reactions. “Histamine liberators” such as tissue hormones, type E immunoglobulins (see p. 300), and drugs can release it. Histamine acts via various types of receptor. Binding to H1 receptors promotes contraction of smoothmuscle in the bronchia, and dilates the capillary vessels and increases their permeability. Via H2 receptors, histamine slows down the heart rate and promotes the formation of HCl in the gastric mucosa. In the brain, histamine acts as a neurotransmitter. Epinephrine is a hormone synthesized in the adrenal glands from tyrosine. Its release is subject to neuronal control.This “emergency hormone” mainly acts on the blood vessels, heart, andmetabolism. It constricts the blood vessels and thereby increases blood pressure; it increases cardiac function; it promotes the degradation of glycogen into glucose in the liver and muscles; and it dilates the bronchia.
Hormones of adrenal glands.
Adrenal glands consist of two parts: external - cortex, internal - medulla.
Each part secrets specific hormones.
Hormones of adrenal medulla – catecholamines (epinephrine, norepinephrine, dopamine).
Chemical structure - these hormones are derivatives of amino acid tyrosine.
Epinephrine, norepinephrine, dopamine exist in
Functions: causes very potent contraction of vessels and increase the blood pressure, increase a pulse rate. Epinephrine relaxes the smooth muscles of bronchi, intestine, promote the contraction of uterus smooth muscle. Epinephrine play a great role in stress reactions.
The effect of epinephrine on carbohydrate metabolism:
- activates the decomposition of glycogen in liver and muscles;
- activates the glycolysis, Krebs cycle and tissue respiration;
- causes the hyperglycemia.
The effect of epinephrine on protein metabolism:
- activates the protein decomposition.
The effect of epinephrine on lipid metabolism:
- activates the tissue lipase, mobilization of lipids and oxidation of fatty acids.
• Hormones, cytokines, interleukins, and growth factors use a variety of signaling mechanisms to facilitate cellular adaptive responses.
• The ligand-receptor complex serves as the initial signal for members of the nuclear receptor family.
• Class II hormones, which bind to cell surface receptors, generate a variety of intracellular signals. These include cAMP, cGMP, Ca2+, phosphatidylinositides, and protein kinase cascades.
• Many hormone responses are accomplished through alterations in the rate of transcription of specific genes.
• The nuclear receptor superfamily of proteins plays a central role in the regulation of gene transcription.
• These receptors, which may have hormones, metabolites, or drugs as ligands, bind to specific DNA elements as homodimers or as heterodimers with RXR. Some—orphan receptors—have no known ligand but bind DNA and influence transcription.
• Another large family of coregulator proteins remodel chromatin, modify other transcription factors, and bridge the nuclear receptors to the basal transcription apparatus.
Hormones of thyroid and parathyroid, structure, mechanism of action.
Thyroid synthesizes two kinds of hormones: iodine containing hormones and calcitonin.
Iodine containing hormones - thyroxine and triiodthyronine.
Thyroxine and triiodthyronine are iodinated derivatives of amino acid tyrosine.
Functions: these hormones are necessary for growth of organism, mental, physical and sex development, they regulate the rate of basal metabolism.
Effect of thyroxine and triiodthyronine on the protein metabolism:
1. in normal concentration stimulate the synthesis of proteins and nucleic acids;
2. in excessive concentration activate the catabolic processes.
Effect of thyroxine and triiodthyronine on the carbohydrate metabolism:
1. promote the absorption of carbohydrates in the intestine;
2. activates the decomposition of glycogen.
Effect of thyroxine and triiodthyronine on the lipid metabolism:
- activate the lipid oxidation and mobilization.
Effect of thyroxine and triiodthyronine on the energy metabolism:
1. activates dehydrogenases of mitochondria;
2. in excessive amount of these hormones the disconnection of tissue respirstion and oxidative phosphorylation takes place. As result the formation of ATP is decreased and the temperature of the organism is increased.
Excessive secretion of thyroid hormones, called hyperthyroidism,
is responsible for Graves' disease,
or exophthalmic goiter
while deficiency in thyroid hormones, hypothyroidism
is characteristic of the disease myxedema.
Calcitonin is synthesized by the parafollicle cells of thyroid.
Chemical structure: peptide.
Functions: - promotes the transition of calcium from blood in bones;
- inhibits the reabsorption of phosphorus in kidneys.
Thus, calcitonin decreases the Ca and P contents in blood.
Parathyroid hormone. Chemical structure: protein.
1. promotes the transition of calcium from bones to blood;
2. promotes the absorption of Ca in the intestine;
3. inhibits the reabsorption of phosphorus in kidneys.
Thus, parathyroid hormone increases the Ca amount in blood and decreases the P amount in blood.
Body Distribution of Calcium and Phosphate
There are three major pools of calcium in the body:
· Intracellular calcium:
· A large majority of calcium within cells is sequestered in mitochondria and endoplasmic reticulum. Intracellular free calcium concentrations fluctuate greatly, from roughly 100 nM to greater than 1 uM, due to release from cellular stores or influx from extracellular fluid. These fluctuations are integral to calcium's role in intracellular signaling, enzyme activation and muscle contractions.
· Calcium in blood and extracellular fluid:
Roughly half of the calcium in blood is bound to proteins. The
concentration of ionized calcium in this compartment is normally almost
invariant at approximately
· Bone calcium:
· A vast majority of body calcium is in bone. Within bone, 99% of the calcium is tied up in the mineral phase, but the remaining 1% is in a pool that can rapidly exchange with extracellular calcium.
As with calcium, the majority of body phosphate (approximately 85%) is present in the mineral phase of bone. The remainder of body phosphate is present in a variety of inorganic and organic compounds distributed within both intracellular and extracellular compartments. Normal blood concentrations of phosphate are very similar to calcium.
Fluxes of Calcium and Phosphate
Maintaining constant concentrations of calcium in blood requires frequent adjustments, which can be described as fluxes of calcium between blood and other body compartments. Three organs participate in supplying calcium to blood and removing it from blood when necessary:
· The small intestine is the site where dietary calcium is absorbed. Importantly, efficient absorption of calcium in the small intestine is dependent on expression of a calcium-binding protein in epithelial cells.
· Bone serves as a vast reservoir of calcium. Stimulating net resorption of bone mineral releases calcium and phosphate into blood, and suppressing this effect allows calcium to be deposited in bone.
The kidney is critcally important in calcium homeostasis. Under normal blood calcium concentrations, almost all of the calcium that enters glomerular filtrate is reabsorbed from the tubular system back into blood, which preserves blood calcium levels. If tubular reabsorption of calcium decreases, calcium is lost by excretion into urine.
Hormonal Control Systems
Maintaining normal blood calcium and phosphorus concentrations is managed through the concerted action of three hormones that control fluxes of calcium in and out of blood and extracellular fluid:
Parathyroid hormone serves to increase blood concentrations of calcium. Mechanistically, parathyroid hormone preserves blood calcium by several major effects:
· Stimulates production of the biologically-active form of vitamin D within the kidney.
· Facilitates mobilization of calcium and phosphate from bone. To prevent detrimental increases in phosphate, parathyroid hormone also has a potent effect on the kidney to eliminate phosphate (phosphaturic effect).
· Maximizes tubular reabsorption of calcium within the kidney. This activity results in minimal losses of calcium in urine.
Four small masses of epithelial tissue are embedded in the connective tissue capsule on the posterior surface of the thyroid glands. These are parathyroid glands, and they secrete parathyroid hormone or parathormone. Parathyroid hormone is the most important regulator of blood calcium levels. The hormone is secreted in response to low blood calcium levels, and its effect is to increase those levels.
Hypoparathyroidism, or insufficient secretion of parathyroid hormone, leads to increased nerve excitability. The low blood calcium levels trigger spontaneous and continuous nerve impulses, which then stimulate muscle contraction.
Since parathyroid gland disease (hyperparathyroidism) was first described in 1925, the symptoms have become known as "moans, groans, stones, and bones...with psychic overtones". Although about 5-7% of people with parathyroid disease (hyperparathyroidism) claim they don't have symptoms and to feel fine when the diagnosis of hyperparathyroidism is made, almost 100% of parathyroid patients will actually say they feel better after the parathyroid problem has been cured--proving they had symptoms. The bottom line: Nearly ALL patients with parathyroid problems have symptoms. Sometimes the symptoms are real obvious, like kidney stones, frequent headaches, and depression. Sometimes the symptoms are not so obvious, like high blood pressure and the inability to concentrate. If you have symptoms, you are almost guaranteed to feel remarkably better once the parathyroid tumor has been removed. As we often tell our parathyroid patients: "you will be amazed at how a 16 minute mini-procedure will change your life!"
About 95 percent of the active thyroid hormone is thyroxine, and most of the remaining 5 percent is triiodothyronine. Both of these require iodine for their synthesis. Thyroid hormone secretion is regulated by a negative feedback mechanism that involves the amount of circulating hormone, hypothalamus, and adenohypophysis.
If there is an iodine deficiency, the thyroid cannot make sufficient hormone. This stimulates the anterior pituitary to secrete thyroid-stimulating hormone, which causes the thyroid gland to increase in size in a vain attempt to produce more hormones. But it cannot produce more hormones because it does not have the necessary raw material, iodine. This type of thyroid enlargement is called simple goiter or iodine deficiency goiter.
Calcitonin is secreted by the parafollicular cells of the thyroid gland. This hormone opposes the action of the parathyroid glands by reducing the calcium level in the blood. If blood calcium becomes too high, calcitonin is secreted until calcium ion levels decrease to normal.
Calcitonin is a hormone that functions to reduce blood calcium levels. It is secreted in response to hypercalcemia and has at least two effects:
· Suppression of renal tubular reabsorption of calcium. In other words, calcitonin enhances excretion of calcium into urine.
· Inhibition of bone resorption, which would minimize fluxes of calcium from bone into blood.
Although calcitonin has significant calcium-lowing effects in some species, it appears to have a minimal influence on blood calcium levels in humans.
Vitamin D acts also to increase blood concentrations of calcium. It is generated through the activity of parathyroid hormone within the kidney. Far and away the most important effect of vitamin D is to facilitate absorption of calcium from the small intestine. In concert with parathyroid hormone, vitamin D also enhances fluxes of calcium out of bone.
Mechanism of steroid hormones action (permeating into the cells):
In difference to hormones of protein and peptide nature, receptors for steroid hormones are located within the cells - in the cytoplasm. From cytoplasm the hormone-receptor complexes is translocated into the nucleus where they interact with DNA of nuclear chromatin causing the activation of genes for respective enzyme proteins. So, if hormones of the first group cause the activation of existing enzyme molecules, the acting on the target cells of steroids and thyroid hormones results in the biosynthesis of new enzyme molecules.
• Receptors for thyroid hormones are members of a large family of nuclear receptors that include those of the steroid hormones. They function as hormone-activated transcription factors and thereby act by modulating gene expression. In contrast to steroid hormone receptors, thyroid hormone receptors bind DNA in the absence of hormone, usually leading to transcriptional repression. Hormone binding is associated with a conformational change in the receptor that causes it to function as a transcriptional activator.
• Currently, four different thyroid hormone receptors are recognized: alpha-1, alpha-2, beta-1 and beta-2.
• The presence of multiple forms of the thyroid hormone receptor, with tissue and stage-dependent differences in their expression, suggests an extraordinary level of complexity in the physiologic effects of thyroid hormone.
• Thyroid hormone receptors bind to short, repeated sequences of DNA called thyroid or T3 response elements (TREs), a type of hormone response element. A TRE is composed of two AGGTCA "half sites" separated by four nucleotides. The half sites of a TRE can be arranged as direct repeats, pallindromes or inverted repeats.
• The DNA-binding domain of the receptor contains two sets of four cysteine residues, and each set chelates a zinc ion, forming loops known as "zinc fingers". A part of the first zinc finger interacts directly with nucleotides in the major groove of TRE DNA, while residues in the second finger interact with nucleotides in the minor groove of the TRE. Thus, the zinc fingers mediate specificity in binding to TREs.
• Thyroid hormone receptors can bind to a TRE as monomers, as homodimers or as heterodimers with the retinoid X receptor (RXR), another member of the nuclear receptor superfamily that binds 9-cis retinoic acid. The heterodimer affords the highest affinity binding, and is thought to represent the major functional form of the receptor.
• Thyroid hormone receptors bind to TRE DNA regardless of whether they are occupied by T3. However, the biological effects of TRE binding by the unoccupied versus the occupied receptor are dramatically different. In general, binding of thyroid hormone receptor alone to DNA leads to repression of transcription, whereas binding of the thyroid hormone-receptor complex activates transcription.
Hormones of adrenal cortex.
Adrenal glands consist of two parts: external - cortex, internal - medulla.
Each part secrets specific hormones.
Hormones synthesized in adrenal cortex are named corticosteroids.
Corticosteroids have potent regulatory effect on all kinds of metabolism. Cholesterol is the precursor of corticosteroids. According to the biological effect corticosteroids are divided on two groups: glucocorticoids and mineralocorticoids. Glucocorticoids regulate the protein, lipid and carbohydrate metabolism, mineralocorticoids - metabolism of water and mineral salt.
The most important glucocorticoids: corticosterone, hydrocortisone, cortisol. The most important mineralocorticoid: aldosterone.
All biological active hormones of adrenal cortex consist of 21 carbon atom and can be reviewed as derivatives of carbohydrate pregnane.
The synthesis of corticosteroids is regulated by ACTH.
In the blood corticosteroids are connected with proteins and transported to different organs.
Time half-life for corticosteroids is about 1 hour.
Ways of metabolism of corticosteroids:
1. Reduction. Corticosteroids accept 4 or 6 hydrogen atoms and form couple compounds with glucuronic acid. These compounds ere excreted by kidneys.
2. Oxidation of 21-st carbon atom.
3. Reduction of ring and decomposition of side chain. As result 17-ketosteroids are formed that are excreted with urine. The determination of 17-ketosteroids in urine - important diagnostic indicator. This is the indicator of adrenal cortex function. In men 17-ketosteroids are also the terminal products of sex hormones metabolism giving important information about testicles function.
4. Corticosteroids can be excreted by kidneys in native structure.
These forms of hormones are lipids. They can enter the cell membrane quite easily and enter right into the nuclei. Steroid hormones are generally carried in the blood bound to specific carrier proteins such as sex hormone binding globulin or corticosteroid binding globulin. Further conversions and catabolism occurs in the liver, other "peripheral" tissues, and in the target tissues.
Because steroids and sterols are lipid soluble, they can diffuse fairly freely from the blood through the cell membrane and into the cytoplasm of target cells. In the cytoplasm the steroid may or may not undergo an enzyme-mediated alteration such as reduction, hydroxylation, or aromatization. In the cytoplasm, the steroid binds to the specific receptor, a large metalloprotein. Upon steroid binding, many kinds of steroid receptor dimerizes, two receptor subunits join together to form one functional DNA-binding unit that can enter the cell nucleus. In some of the hormone systems known, the receptor is associated with a heat shock protein which is released on the binding of the ligand, the hormone. Once in the nucleus, the steroid-receptor ligand complex binds to specific DNA sequences and induces transcription of its target genes.
It discussing steroid hormones, one is required to talk about cholesterol; cholesterol is know as a sterol, which is a natural product from the steroid nucleus. Cholesterol is very important, as we learned, in the production of steroid hormones, in fact they are the precursor for bile acids (bile acids aid in fat digestion), steroid hormones, and provitamin D (When irradiated by sunlight it changes to vitamin D3.
Cholesterol, if we recall, is incorporated into the cell membrane by lipoproteins.There it plays a role in the regulation of membrane fluidity. It has been stated that cholesterol is probably responsible for permitting steroid hormones to enter the cell.
Synthesis of steroid hormons
Functions of glucocorticoids.
Glucocorticoids have antiinflammatory, antiallergic, antiimmune effect. They support the blood pressure and constancy of the extracellular liquids.
The effect of glucocorticoids on protein metabolism:
1. stimulate the catabolic processes (protein decomposition) in connective, lymphoid and muscle tissues and activate the processes of protein synthesis in liver;
2. stimulate the activity of aminotransferases;
3. activate the synthesis of urea.
The effect of glucocorticoids on carbohydrate metabolism:
1. activate the gluconeogenesis;
2. inhibit the activity of hexokinase;
3. activate the glycogen synthesis in liver.
Glucocorticoids causes the hyperglycemia.
The effect of glucocorticoids on lipid metabolism:
1. promote the absorption of lipids in intestine;
2. activate lipolisis;
3. activate the conversion of fatty acids in carbohydrates.
Functions of mineralocorticoids.
Secretion of mineralocorticoids is regulated by renin-angiotensine system
- activates the reabsorption of Na+, Cl- and water in kidney canaliculuses;
- promote the excretion of K+ by kidneys, skin and saliva.
Deficiency of corticosteroids causes Addison's disease.
For this disease the hyperpigmentation is typical because the deficiency of corticosteroids results in the excessive synthesis of ACTH.
Hyperfunction of adrenal cortex causes Icenko-Kushing syndrome. This state is called steroid diabetes. Symptoms: hyperglycemia, glucosuria, hypercholesterolemia, hypernatriemia, hyperchloremia, hypokaliemia.
Adrenal cortex hormones and their artificial analogs are often used in clinic: for treatment of allergic and autoimmune diseases, in hard shock states.
Blood and urine cortisol, together with the determination of adrenocorticotropic hormone (ACTH), are the three most important tests in the investigation of Cushing's syndrome (caused by an overproduction of cortisol) and Addison's disease (caused by the underproduction of cortisol).
Reference ranges for cortisol vary from laboratory to laboratory but are usually within the following ranges for blood:
· adults (): 6-28 mg/dL; adults (): 2-12 mg/dL
· child one to six years (): 3-21 mg/dL; child one to six years (): 3-10 mg/dL
· newborn: 1/24 mg/dL.
Reference ranges for cortisol vary from laboratory to laboratory, but are usually within the following ranges for 24-hour urine collection:
· adult: 10-100 mg/24 hours
· adolescent: 5-55 mg/24 hours
· Child: 2-27 mg/24 hours.
Increased levels of cortisol are found in Cushing's syndrome, excess thyroid (hyperthyroidism), obesity, ACTH-producing tumors, and high levels of stress.
Decreased levels of cortisol are found in Addison's disease, conditions of low thyroid, and hypopituitarism, in which pituitary activity is diminished.
A rare disorder in which symptoms are caused by a deficiency of hydrocortisone (cortisol) and aldosterone, two corticosteroid hormones normally produced by a part of the adrenal glands called the adrenal cortex. Symptoms include weakness, tiredness, vague abdominal pain, weight loss, skin pigmentation and low blood pressure.
A hormonal disorder caused by an abnormally high level of corticosteroid hormones. Symptoms include high blood sugar levels, a moon face, weight gain, and increased blood pressure
Since cortisol production by the adrenal glands is normally under the control of the pituitary (like the thyroid gland), overproduction can be caused by a tumor in the pituitary or within the adrenal glands themselves. When a pituitary tumor secretes too much ACTH (Adrenal Cortical Tropic Hormone), it simply causes the otherwise normal adrenal glands to produce too much cortisol. This type of Cushings syndrome is termed "Cushings Disease" and it is diagnosed like other endocrine disorders by measuring the appropriateness of hormone production. In this case, serum cortisol will be elevated, and, serum ACTH will be elevated at the same time.
When the adrenal glands develop a tumor, like any other endocrine gland, they usually produce excess amounts of the hormone normally produced by these cells. If the adrenal tumor is composed of cortisol producing cells, excess cortisol will be produced which can be measured in the blood. Under these conditions, the normal pituitary will sense the excess cortisol and will stop making ACTH in an attempt to slow the adrenal down. In this manner, physicians can readily distinguish whether excess cortisol is the result of a pituitary tumor, or an adrenal tumor.
Even more rare (but placed here for completion sake) is when excess ACTH is produced somewhere other than the pituitary. This is extremely uncommon, but certain lung cancers can make ACTH (we don't know why) and the patients develop Cushings Syndrome in the same way they do as if the ACTH was coming from the pituitary.
Causes of Cushings Syndrome
ACTH Dependent (80%)
Pituitary Tumors (60%)
Lung Cancers (5%)
ACTH Independent (20%)
Benign Adrenal Tumors (adenoma) (25%)
Malignant Adrenal Tumors (adrenal cell carcinoma) (10%)
Testing for Cushings Syndrome
The most sensitive test to check for the possibility of this disease is to measure the amount of cortisol
excreted in the during during a 24 hour time period. Cortisol is normally secreted in different amounts during the day and night, so this test usually will be repeated once or twice to eliminate the variability which is normally seen. This normal variability is why simply checking the amount of cortisol in the blood is not a very reliable test. A 24 hour free cortisol level greater than 100 ug is diagnostic of Cushings syndrome. The second test which helps confirms this diagnosis is the suppression test which measures the cortisol secretion following the administration of a powerful synthetic steroid which will shut down steroid production in everybody with a normal adrenal gland. Subsequent tests will distinguish whether the disease is due to an ACTH dependent or independent cause.
Invariably, once the diagnosis is made, patients will undergo a CT scan (or possibly an MRI or Ultrasound) of the adrenal glands to look for tumors in one or both of them (more information on adrenal x-ray tests on another page). If the laboratory test suggest a pituitary origin, a CT or MRI of the brain (and possibly of the chest as well) will be performed.
Treatment of Cushings Syndrome
Obviously, the treatment of this
disease depends upon the cause. Pituitary tumors are usually removed surgically
and often treated with radiation therapy. Neurosurgeons and some ENT surgeons
specialize in these tumors. If the cause is determined to be within a single
adrenal gland, this is treated by surgical removal. If the tumor has
characteristics of cancer on any of the x-ray tests, then a larger,
conventional operation is in order. If a single adrenal gland possesses a
small, well defined tumor, it can usually be removed by the new technique of
Glucocorticoids originate in the adrenal cortex and affect mainly metabolism in diverse ways; decrease inflammation and increase resistance to stress.
Mineralocorticoids originate in adrenal cortex and maintain salt and water balance.
Estrogens originate in the adrenal cortex and gonads and primarily affect maturation and function of secondary sex organs (female sexual determination).
Androgens originate in the adrenal cortex and gonads and primarily affect maturation and function of secondary sex organs (male sexual determination).
Progestins originate from both ovaries and placenta, and mediate menstrual cycle and maintain pregnancy.
Androgens and estrogens play a major role in the development of both sexes secondary characteristics. Androgens, or testosterone and androsterone give the male its sex characteristics during puberty and for promoting tissue and muscle growth. Estrogens, or estrone and estradiol are forms of testosterone synthesized in the ovaries, which control female secondary characteristics and regulation of the menstrual cycle. Another sex hormone is needed for preparing the uterus for implantation of the ovum, this hormone is progesterone.
Hormones are needed throughout the body for various functions, however, just as important as these function is the regulation and control of these steroids.
Sex hormones are synthesized in testicles, ovaries. Smaller amount of sex hormones are produced in adrenal cortex and placenta. Small amount of male sex hormones are produced in ovaries and female sex hormones - in testicles.
Male sex hormones are called androgens and female - estrogens.
Chemical structure - steroids.
Synthesis and secretion of the sex hormones are controlled by the pituitary honadotropic hormones. Sex hormones act by means of the activation of gene apparatus of cells. Catabolism of sex hormones takes place in liver. The time half-life is 70-90 min.
The main estrogens: estradiol, estrole, estriole (are produced by follicles) and progesterone (is produced by yellow body and placenta). The main biological role of estrogens - conditioning for the reproductive female function (possibility of ovum fertilization). Estradiol results in the proliferation of endometrium and progesterone stimulates the conversion of endometrium in decidual tissue which is ready for ovum implantation. Estrogens also cause the development of secondary sexual features.
The main androgen is testosterone. Its synthesis is regulated by the luteinizing hormone. Testosterone forms the secondary sexual features in males.
Effect of sex hormones on protein metabolism:
1. stimulate the processes of protein, DNA, RNA synthesis;
2. cause the positive nitrogenous equilibrium.
Effect of sex hormones on carbohydrate metabolism:
1. activate the Krebs cycle;
2. activate the synthesis of glycogen in liver.
Effect of sex hormones on lipid metabolism:
1. enhance the oxidation of lipids;
2. inhibit the synthesis of cholesterol.
Effect of sex hormones on energy metabolism:
- stimulate the Krebs cycle, tissue respiration and ATP production.
Sex hormones are used for treatment of variety diseases. For example, testosterone and its analogs are used as anabolic remedies; male sex hormones are used for the treatment of malignant tumor of female sex organs and vice versa.
Prostaglandins. The precursor of prostaglandins is arachidonic acid. Time half-life - 30 s. There are different prostaglandins and they have a lot of physiological and pharmacological effects and different prostaglandins have different effects.
Prostaglandins: - decrease the activity of lipid tissue lipase;
- regulate the calcium metabolism in muscle tissue and as result effect on the contraction and relaxation of muscles;
- inhibit the gastric secretion;
- stimulate the formation of steroids.
Kallicrein-kinin system. Kinins - group of peptides with similar structure and biological properties. The main kinins - bradykinin and kallidine.
Kinins are formed from their precursors kininogens that are synthesized in liver owing to acting of kallicreins. Kallicreins are also formed from inactive precursors prekallicreins by means of proteolysis.
Functions: - kinins relax the smooth muscles of blood vessels and decrease the blood pressure;
- increase the capillaries permeability;
- takes part in the inflammatory processes.
Renin-angiotensin system. Renin - enzyme that is synthesized in special cells located near the renal glomerules.
Renin acts on angiotensinogen. As result angiotensin-I is formed. Under the effect of peptidase angiotensin-I is converted to angiotensin-II. Angiotensin-II causes 2 effects:
- narrows the vessels and increases the blood pressure;
- stimulates the secretion of aldosterone.
The decrease of renal blood stream is the specific stimulant for renin secretion.
Energy releasing processes, ones that "generate" energy, are termed exergonic reactions. Reactions that require energy to initiate the reaction are known as endergonic reactions. All natural processes tend to proceed in such a direction that the disorder or randomness of the universe increases (the second law of thermodynamics).
Time-energy graphs of an exergonic reaction (top) and endergonic reaction (bottom). Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
Oxidation/Reduction | Back to Top
Biochemical reactions in living organisms are essentially energy transfers. Often they occur together, "linked", in what are referred to as oxidation/reduction reactions. Reduction is the gain of an electron. Sometimes we also have H ions along for the ride, so reduction also becomes the gain of H. Oxidation is the loss of an electron (or hydrogen). In oxidation/reduction reactions, one chemical is oxidized, and its electrons are passed (like a hot potato) to another (reduced, then) chemical. Such coupled reactions are referred to as redox reactions. The metabolic processes glycolysis, Kreb's Cycle, and Electron Transport Phosphorylation involve the transfer of electrons (at varying energy states) by redox reactions.
Passage of electrons from compound A to compound B. When A loses its electrons it is oxidized; when B gains the electrons it is reduced. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
Oxidation/reduction via an intermediary (energy carrier) compound, in this case NAD+. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
Catabolism and Anabolism | Back to Top
is the total series of chemical reactions involved in synthesis of organic
must be able to manufacture (synthesize) all the organic compounds they need. Heterotrophs
can obtain some of their compounds in their diet (along with their energy). For
example humans can synthesize 12 of the 20 amino acids,
we must obtain the other
Enzymes: Organic Catalysts | Back to Top
Enzymes allow many chemical reactions to occur within the homeostasis constraints of a living system. Enzymes function as organic catalysts. A catalyst is a chemical involved in, but not changed by, a chemical reaction. Many enzymes function by lowering the activation energy of reactions. By bringing the reactants closer together, chemical bonds may be weakened and reactions will proceed faster than without the catalyst.
The use of enzymes can lower the activation energy of a reaction (Ea). Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
Enzymes can act rapidly, as in the case of carbonic anhydrase (enzymes typically end in the -ase suffix), which causes the chemicals to react 107 times faster than without the enzyme present. Carbonic anhydrase speeds up the transfer of carbon dioxide from cells to the blood. There are over 2000 known enzymes, each of which is involved with one specific chemical reaction. Enzymes are substrate specific. The enzyme peptidase (which breaks peptide bonds in proteins) will not work on starch (which is broken down by human-produced amylase in the mouth).
Enzymes are proteins. The functioning of the enzyme is determined by the shape of the protein. The arrangement of molecules on the enzyme produces an area known as the active site within which the specific substrate(s) will "fit". It recognizes, confines and orients the substrate in a particular direction.
Space filling model of an enzyme working on glucose. Note the shape change in the enzyme (indicated by the red arrows) after glucose has fit into the binding or active site. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
The induced fit hypothesis suggests that the binding of the substrate to the enzyme alters the structure of the enzyme, placing some strain on the substrate and further facilitating the reaction. Cofactors are nonproteins essential for enzyme activity. Ions such as K+ and Ca+2 are cofactors. Coenzymes are nonprotein organic molecules bound to enzymes near the active site. NAD (nicotinamide adenine dinucleotide).
A cartoonish view of the formation of an enzyme-substrate complex. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
Enzymatic pathways form as a result of the common occurrence of a series of dependent chemical reactions. In one example, the end product depends on the successful completion of five reactions, each mediated by a specific enzyme. The enzymes in a series can be located adjacent to each other (in an organelle or in the membrane of an organelle), thus speeding the reaction process. Also, intermediate products tend not to accumulate, making the process more efficient. By removing intermediates (and by inference end products) from the reactive pathway, equilibrium (the tendency of reactions to reverse when concentrations of the products build up to a certain level) effects are minimized, since equilibrium is not attained, and so the reactions will proceed in the "preferred" direction.
Negative feedback and a metabolic pathway. The production of the end product (G) in sufficient quantity to fill the square feedback slot in the enzyme will turn off this pathway between step C and D. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
Temperature: Increases in temperature will speed up the rate of nonenzyme mediated reactions, and so temperature increase speeds up enzyme mediated reactions, but only to a point. When heated too much, enzymes (since they are proteins dependent on their shape) become denatured. When the temperature drops, the enzyme regains its shape. Thermolabile enzymes, such as those responsible for the color distribution in Siamese cats and color camouflage of the Arctic fox, work better (or work at all) at lower temperatures.
Concentration of substrate and product also control the rate of reaction, providing a biofeedback mechanism.
Activation, as in the case of chymotrypsin, protects a cell from the hazards or damage the enzyme might cause.
Changes in pH will also denature the enzyme by changing the shape of the enzyme. Enzymes are also adapted to operate at a specific pH or pH range.
Plot of enzyme activity as a function of pH for several enzymes. Note that each enzyme has a range of pH at which it is active as well as an optimal pH at which it is most active. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
Allosteric Interactions may allow an enzyme to be temporarily inactivated. Binding of an allosteric effector changes the shape of the enzyme, inactivating it while the effector is still bound. Such a mechanism is commonly employed in feedback inhibition. Often one of the products, either an end or near-end product act as an allosteric effector, blocking or shunting the pathway.
Action of an allosteric inhibitor as a negative control on the action of an enzyme. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
Competitive Inhibition works by the competition of the regulatory compound and substrate for the binding site. If enough regulatory compound molecules bind to enough enzymes, the pathway is shut down or at least slowed down. PABA, a chemical essential to a bacteria that infects animals, resembles a drug, sulfanilamide, that competes with PABA, shutting down an essential bacterial (but not animal) pathway.
Top: general diagram showing competitor in the active site normally occupied by the natural substrate; Bottom: specific case of succinate dehydrogenase and its natural substrate (succinate) and competitors (oxalate et al.). Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
Noncompetitive Inhibition occurs when the inhibitory chemical, which does not have to resemble the substrate, binds to the enzyme other than at the active site. Lead binds to SH groups in this fashion. Irreversible Inhibition occurs when the chemical either permanently binds to or massively denatures the enzyme so that the tertiary structure cannot be restored. Nerve gas permanently blocks pathways involved in nerve message transmission, resulting in death. Penicillin, the first of the "wonder drug" antibiotics, permanently blocks the pathways certain bacteria use to assemble their cell wall components.
Learning Objectives | Back to Top
Reactions that show a net loss in energy are said to be exergonic; reactions that show a net gain in energy are said to be endergonic. Describe an example of each type of chemical reaction from everyday life.
What is meant by a reversible reaction? How might this be significant to living systems?
What are enzymes? Explain their importance.
Explain what happens when enzymes react with substrates.
Links | Back to Top
Biology Project (U of A) Energy and Enzymes Problem Set
The G6PD Deficiency Homepage 400 Million folks have this problem, and it is enzymatic!
Enzyme Inhibition and Regulation (3/1/96) From WSU's chemistry site.
Enzyme Reaction Tutorial (U.C. Davis)
EC Enzyme Search the EC enzyme databse, includes links to OMIM (Online Mendelain Inheritance in Man) and SWISSPROT (Swiss Protein Database).
Interactive Cytochrome Oxidase You will need the Chime plugin (available at this site), but it will be well worth it. View either of the subunits of cytochrome oxidase as well as related molecules. You can check buttons on the left frame to display selected portions of the molecule, zoom in, and zoom out.
Metabolic Pathways of Biochemistry Check out the metabolic pathway of your choice in 2-D or 3-D (with the Chime plugin) models. is provided than in most general biology textbooks, but the point is driven home...a chemical reaction in one of these pathways needs its own enzyme.
Mitochondria (Mitochondria Under Microscope)
are thought to have evolved at least 1.8 billion years ago from primitive
bacteria which enjoyed such a symbiotic relationship with early eukaryotic
cells. Mitochondria still show some signs of their ancient origin.
Mitochondrial ribosome's are the 70S (bacterial) type,
in contrast to the 80S ribosome's found elsewhere in the cell. As in
prokaryotes there is a very high proportion of coding DNA, and an absence of
repeats. Mitochondria genes are transcribed as multigenic transcripts which are
cleaved and polyadenylated to yield mature mRNAs. Unlike their nuclear cousins,
mitochondrial genes are small, generally lacking introns, and the chromosomes
are circular, conforming to the bacterial pattern.
Observed in 1850 by Kollicker in Muscle Cell. Later Identified as the center for aerobicareobic respiration in the cell.
(Shows how a mitochondria is used in aerobic respiration)
(Shows the inter and outer structure of a mitochondria)
(Shows the outer and inter walls of a mitochondria in great detail)
Mitochondria contain two membranes, separated by a space. Both are the typical "unit membrane" (railroad track) in structure. Inside the space enclosed by the inner membrane is the matrix. This appears moderately dense and one may find strands of DNA, ribosomes, or small granules in the matrix. The mitochondria are able to code for part of their proteins with these molecular tools. The above cartoon shows the diagram of the mitochondrial membranes and the enclosed compartments.
(Shows the energy cycles that take place in the cell)
Cellular respiration is the process of oxidizing food molecules, like glucose, to carbon dioxide and water. The energy released is trapped in the form of ATP for use by all the energy consuming activities of the cell. The process occurs in two phases:
- glycolysis, the breakdown of glucose to pyruvic acid
- the complete oxidation of pyruvic acid to carbon dioxide and water
The energy conversion is as follows:
C6H12O6 + 6O<2 -> 6CO2 + 6H2O + energy (ATP)
There are two important ways a cell can harvest energy from food: fermentation and cellular respiration. Both start with the same first step: the process of glycolysis which is the breakdown or splitting of glucose (6 carbons) into two 3-carbon molecules called pyruvic acid. The energy from other sugars, such as fructose, is also harvested using this process. Glycolysis is probably the oldest known way of producing ATP. There is evidence that the process of glycolysis predates the existence of O2 in the Earth’s atmosphere and organelles in cells:
Glycolysis does not need oxygen as part of any of its chemical reactions. It serves as a first step in a variety of both aerobic and anaerobic energy-harvesting reactions.
Glycolysis happens in the cytoplasm of cells, not in some specialized organelle.
Glycolysis is the one metabolic pathway found in all living organisms.
2 pyruvic acid molecules
+ 4 H+ + energy stored in 2 ATP molecules
In fermentation these pyruvic acid molecules are turned into some “waste” product, and a little bit of energy (only two ATP molecules per molecule of glucose – actually four are produced in glycolysis, but two are used up) is produced. Out of many possible types of fermentation processes, two of the most common types are lactic acid fermentation and alcohol fermentation.
Pyruvic Acid + 2 H+
Lactic Acid Ethanol
Lactic acid fermentation is done by some fungi, some bacteria like the Lactobacillus acidophilus. in yogurt, and sometimes by our muscles. Normally our muscles do cellular respiration like the rest of our bodies, using O2 supplied by our lungs and blood. However, under greater exertion when the oxygen supplied by the lungs and blood system can’t get there fast enough to keep up with the muscles’ needs, our muscles can switch over and do lactic acid fermentation. In the process of lactic acid fermentation, the 3-carbon pyruvic acid molecules are turned into lactic acid. It is the presence of lactic acid in yogurt that gives it its sour taste, and it is the presence of lactic acid in our muscles “the morning after” that makes them so sore. Once our muscles form lactic acid, they can’t do anything else with it, so until it is gradually washed away by the blood stream and carried to the liver (which is able to get rid of it), our over-exerted muscles feel stiff and sore even if they haven’t been physically injured.
Alcohol fermentation is done by yeast and some kinds of bacteria. The “waste” products of this process are ethanol and carbon dioxide (CO2). Humans have long taken advantage of this process in making bread, beer, and wine. In bread making, it is the CO2 which forms and is trapped between the gluten (a long protein in wheat) molecules that causes the bread to rise, and the ethanol (often abbreviated as EtOH – do you remember how to draw it?) evaporating that gives it its wonderful smell while baking. The effects of the ethanol in beer and wine are something with which many college students are familiar (sometimes too familiar?), and it is the CO2 produced by the process of fermentation that makes these beverages effervescent.
Dr. Fankhauser has a number of fermentation-related recipes online, complete with photographs:
His main cheese page
A recipe for cheese using one gallon of milk
A recipe for cheese using five gallons of milk
Homemade root beer
Homemade ginger ale
A recipe for whole wheat bread
General information on milk-fermenting bacteria
An analogy can be drawn between the process of cellular respiration in our cells and a car. The mitochondria are the engines of our cells where sugar is burned for fuel and the exhaust is CO2 and H2O. Note that in a car that burned fuel perfectly, the only exhaust should theoretically be CO2 and H2O also.
In contrast to fermentation, in the process of cellular respiration, the pyruvic acid molecules are broken down completely to CO2 and more energy released. Note that three molecules of O2 must react with each molecule of pyruvic acid to form the three carbon dioxide molecules, and three molecules of water are also formed to “use up” the hydrogens. As mentioned above, in glycolysis, a total of four molecules of ATP are produced, but two are used up in other steps in the process. Additional ATP is produced during the Krebs Cycle and the Electron Transport Chain, resulting in a grand total of 40 ATP molecules produced from the breakdown of one molecule of glucose via cellular respiration. Since two of those are used up during glycolysis, in prokaryotes a net total of 38 molecules of ATP are produced by cellular respiration. Most prokaryotes have very simple cells which lack several types of organelles present in eukaryotes, and therefore the Krebs Cycle and the Electron Transport Chain occur in the cytoplasm and/or using chemicals embedded in the cell membrane. In contrast, eukaryotes have more complex cells with more specialized organelles to perform given functions. In eukaryotes, the Krebs Cycle and Electron Transport Chain occur within the mitochondria, and thus the pyruvic acid resulting from glycolysis must be sent into the mitochondria for these reactions to occur. However, to move one molecule of pyruvic acid (remember each molecule of glucose turns into two pyruvic acid molecules) from the cytoplasm into a mitochondrion “costs” the cell one molecule of ATP (therefore two ATPs for a whole glucose), thus a net total of 36 ATP molecules per molecule of glucose is produced in eukaryotes as compared to only two in fermentation. The overall reaction for cellular respiration is C6H12O6 + 6O2 6CO2 + 6H2O (+ energy for the cell to use for other things).
Pyruvic Acid + 2 H+ + 3 O2
3 Carbon Dioxide + 3 H2O+ 34 ATP
In glycolysis and the Krebs cycle, there are also a number of electrons released as the glucose molecule is broken down. The cell must deal with these electrons in some way, so they are stored by the cell by forming a compound called NADH by the chemical reaction, NAD+ + H+ + 2e– NADH. This NADH is used to carry the electrons to the electron transport chain, where more energy is harvested from them.
In eukaryotes, the pyruvic acid from glycolysis must be transferred into the mitochondria to be sent through the Krebs cycle, also known as the citric acid cycle, at a “cost” of one ATP per molecule of pyruvic acid. In this cycle, discovered by Hans Krebs, the pyruvic acid molecules are converted to CO2, and two more ATP molecules are produced per molecule of glucose. First, each 3-carbon pyruvic acid molecule has a CO2 broken off and the other two carbons are transferred to a molecule called acetyl coenzyme A, while a molecule of NADH is formed from NAD+ for each pyruvic acid (= 2 for the whole glucose). These acetyl CoA molecules are put into the actual cycle, and after the coenzyme A part is released, eventually each 2-carbon piece is broken apart into two molecules of CO2. In the process, for each acetyl CoA that goes into the cycle, three molecules of NADH, one molecule of FADH2, and one molecule of ATP are formed (= 6 NADH, 2 FADH2, and 2 ATP per whole glucose).
The electron transport chain is a system of electron carriers embedded into the inner membrane of a mitochondrion. As electrons are passed from one compound to the next in the chain, their energy is harvested and stored by forming ATP. For each molecule of NADH which puts its two electrons in, approximately three molecules of ATP are formed, and for each molecule of FADH2, about two molecules of ATP are formed.
Many of the compounds that make up the electron transport chain belong to a special group of chemicals called cytochromes. The central structure of a cytochrome is a porphyrin ring like chlorophyll but with iron in the center (chlorophyll has magnesium). A porphyrin with iron in the center is called a heme group, and these are also found in hemoglobin in our blood.
At the last step in the electron transport chain, the “used up” electrons, along with some “spare” hydrogen ions are combined with O2 (we finally got around to the O2) to form water as a waste product: 4e- + 4H+ + O2 2H2O.
Click on the heme group to see how to draw one.
Click the picture to re-start or press [ESC] to stop. You may also “write” on the picture. Unfortunately, Corel only has a Plug-In for Win 95/NT, so this won’t work with Win 3.1 or Mac.
Many of the enzymes in the cells of organisms need other helpers to function. These non-protein enzyme helpers are called cofactors and can include substances like iron, zinc, or copper. If a cofactor is an organic molecule, it then is called a coenzyme. Many of the vitamins needed by our bodies are used as coenzymes to help our enzymes to do their jobs. Vitamin B1 (thiamine) is a coenzyme used in removing CO2 from various organic compounds. B2 (riboflavin) is a component of FAD (or FADH2), one of the chemicals used to transport electrons from the Krebs cycle to the electron transport chain. Vitamin B3 (niacin) is a component of NAD+ (or NADH) which is the major transporter of electrons from glycolysis and the Krebs cycle to the electron transport chain. Without enough of these B vitamins, our ability to get the energy out of our food would come to a grinding halt! B6 (pyridoxine), B12 (cobalamin), pantothenic acid, folic acid, and biotin are all other B vitamins which serve as coenzymes at various points in metabolizing our food. Interestingly, B12 has cobalt in it, a mineral which we need in only very minute quantities, but whose absence can cause symptoms of deficiency.
My mother once had a friend who had porphyria, a dominant genetic disorder in which the person’s body cannot properly make porphyrin rings. This would, thus, affect the person’s ability to make both hemoglobin to carry oxygen in the blood and cytochromes for the electron transport chain. This woman’s symptoms were quite variable. At times, she would appear nearly normal while on other occasions she would have to be hospitalized for temporary paralysis of part of her body or other symptoms. There were a number of foods and drugs she had to avoid because they would trigger or worsen her symptoms. She frequently was in a lot of pain. Because porphyria is a dominant genetic disorder, there was a 50% chance this woman’s daughter would also have porphyria. Thus after the woman was diagnosed with porphyria, a number of tests were also run on the girl, and she was more carefully monitored as she grew up. My mother eventually lost contact with them, so I never heard the end of the story.
Because there are a number of enzymes and steps involved in forming porphyrin rings, there are a number of possible points in the process where genetic defects could occur. The Merck Manual says there are eight steps in the process of making porphyrin rings, with genetic abnormalities possible in seven of the eight enzymes.
years ago, Dr. Fankhauser mentioned to me that he heard somewhere that an
First, let’s assume that person eats an “average” dietary intake of 2500 KCal of food energy (a number listed on the side of many food packages and a reasonable amount that such a person might consume).
just out of curiosity, let’s assume that all (100%) of that is glucose (In real
life, that would be a terrible idea! We need all the other nutrients that we
get from eating a variety of foods.). Since carbohydrates store about 4 KCal of
energy per gram, that would mean that 2500 KCal of glucose would be equivalent
Also, just for the sake of argument, let’s assume that 100% of the ingested glucose is burned for fuel, and that the process is 100% efficient so there is no waste (in real life, our bodies would never use all 100% for fuel – some gets used to build other chemicals, and just like the fuel efficiency in our automobiles, the process is never 100% efficient.). Since, as was mentioned above, eukaryotes make about 36 moles of ATP from every mole of glucose, then those 3.74 moles of glucose would be equivalent to 125 moles of ATP.
molecular weight of
ATP is 507 g/m, so that would be
if it was really possible to meet all of those background assumptions and a
As another example:
suppose a person would consume one 12-oz. can of soft drink,
most types of soft drink contain
about 41 to
suppose all of that sugar would be glucose,
suppose the person’s body burns all of that sugar for fuel and does not store any of it as fat or use any of it in other ways, and
suppose the process of cellular respiration is 100% efficient and the sugar is completely oxidized to CO2 and H2O.
since the molecular weight of
glucose is 180g/m, the
since cellular respiration produces
since the MW of ATP
is 507 g/m, that would be equivalent to
Recently I received an e-mail message from a student who asked how long the whole process takes. While I have never seen any information on that in print, a rough approximation can also be calculated from the above statistic:
If, as mentioned above, an
40 kg/24 hr Ч 1 hr/60 min Ч 1000 g/kg = about 27.8 g/min.
Since the molecular weight of
ATP is 507 g/m, then
that 27.8 g/min Ч 1 m/507 g = 0.0548 m/min.
Avagadro’s number says that
there are always 6.02 x 1023 molecules/mole,
so 0.0548 m/min Ч 6.02 x 1023 molecules/mole = 3.30 x 1022 molecules/min.
since there are 60 sec/min, then that’s
3.30 x 1022 molecules/min Ч 1min/60 sec = 5.50 x 1020 molecules/sec made by a
so that would be equivalent to
5.50 x 1020 molecules/sec ч
or Ч 1kg/1000 g = 7.85 Ч 1015 molecules/sec/g of body
or Ч 1g/1000 mg = 7.85 Ч 1012 molecules/sec/mg of body
or Ч 1mg/1000 µg = 7.85 Ч 109 molecules/sec/µg of body.
The pyruvate molecules produced during glycolysis contains a lot of energy in the bonds between their molecules. In order to use that energy, the cell must convert it into the form of ATP. To do so, pyruvate molecules are processed through the Kreb Cycle, also known as the citric acid cycle.
(Kerbs Cycle as a drawing)
1. Prior to entering the Krebs Cycle, pyruvate must be converted into acetyl CoA. This is achieved by removing a CO2 molecule from pyruvate and then removing an electron to reduce an NAD+ into NADH. An enzyme called coenzyme A is combined with the remaini ow:
2. Citrate is formed when the acetyl group from acetyl CoA combines with oxaloacetate from the previous Krebs cycle..
3. Citrate is converted into its isomer isocitrate..
4. Isocitrate is oxidized to form the 5-carbon α-ketoglutarate. This step releases one molecule of CO2 and reduces NAD+ to NADH2+.
5. The α-ketoglutarate is oxidized to succinyl CoA, yielding CO2 and NADH2+.
6. Succinyl CoA releases coenzyme A and phosphorylates ADP into ATP.
7. Succinate is oxidized to fumarate, converting FAD to FADH2.
8. Fumarate is hydrolized to form malate.
9. Malate is oxidized to oxaloacetate, reducing NAD+ to NADH2+.
We are now back at the beginning of the Krebs Cycle. Because glycolysis produces two pyruvate molecules from one glucose, each glucose is processes through the kreb cycle twice. For each molecule of glucose, six NADH2+, two FADH2, and two ATP.
These two pictures show the Electron Transport Chain
The schematic diagram above illustrates a mitochondrion. In the animation, watch as NADH transfers H+ ions and electrons into the electron transport system.
1. Protons are translocated across the membrane, from the matrix to the intermembrane space
2. Electrons are transported along the membrane, through a series of protein carriers
3. Oxygen is the terminal electron acceptor, combining with electrons and H+ ions to produce water
delivers more H+ and electrons into the ETS, the proton gradient
increases, with H+ building up outside the inner mitochondrial
replicate like bacterial cells. When they get too large they undergo
fission. This involves a furrowing of the inner and the outer membrane as
if someone was pinching the mitochondria. The two daughter mitochondria
split. The Mitochondria must first replicate their DNA.
Citric acid cycle
From Wikipedia, the free encyclopedia
Overview of the citric acid cycle
The citric acid cycle (also known as the tricarboxylic acid cycle, the TCA cycle, or the Krebs cycle, after Hans Adolf Krebs who identified the cycle) is a series of chemical reactions of central importance in all living cells that use oxygen as part of cellular respiration. In aerobic organisms, the citric acid cycle is part of a metabolic pathway involved in the chemical conversion of carbohydrates, fats and proteins into carbon dioxide and water to generate a form of usable energy. It is the third of four metabolic pathways that are involved in carbohydrate catabolism and ATP production, the other three being glycolysis and pyruvate oxidation before it, and respiratory chain after it.
- The citric acid cycle begins with Acetyl-CoA transferring its two-carbon acetyl group to the four-carbon acceptor compound, oxaloacetate, forming citrate, a six-carbon compound.
- The citrate then goes through a series of chemical transformations, losing first one, then a second carboxyl group as CO2.
- Most of the energy made available by the oxidative steps of the cycle is transferred as energy-rich electrons to NAD+, forming NADH. For each acetyl group that enters the citric acid cycle, three molecules of NADH are produced.
- Electrons are also transferred to the electron acceptor FAD, forming FADH2.
- At the end of each cycle, the four-carbon oxaloacetate has been regenerated, and the cycle continues. Products of the first turn of the cycle are one GTP, three NADH, one FADH2, and two CO2.
- Because two acetyl-CoA molecules are produced from each glucose molecule, two cycles are required per glucose molecule.
- At the end of all cycles, the products are two GTP, six NADH, two FADH2, four CO2.
The sum of all reactions in the citric acid cycle is:
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O → CoA-SH + 3 NADH + 3 H+ + FADH2 + GTP + 2 CO2
(the above reaction is equilibrated if Pi represents the H2PO4- ion, GDP the GDP2- ion and GTP the GTP3- ion).
Two carbons are oxidized to CO2, and the energy from these reactions is stored in GTP, NADH and FADH2. NADH and FADH2 are coenzymes (molecules that enable or enhance enzymes) that store energy and are utilized in oxidative phosphorylation.
Acetyl CoA +
NADH + H+
NADH + H+
NADH + H+
A simplified view of the process
- The citric acid cycle begins with Acetyl-CoA transferring its two-carbon acetyl group to the four-carbon acceptor compound, oxaloacetate, forming citrate, a six-carbon compound.
- The citrate then goes through a series of chemical transformations, losing first one, then a second carboxyl group as CO2.
- Most of the energy made available by the oxidative steps of the cycle is transferred as energy-rich electrons to NAD+, forming NADH. For each acetyl group that enters the citric acid cycle, three molecules of NADH are produced.
- Electrons are also transferred to the electron acceptor FAD, forming FADH2.
- At the end of each cycle, the four-carbon oxaloacetate has been regenerated, and the cycle continues. Products of the first turn of the cycle are one GTP, three NADH, one FADH2, and two CO2.
- Because two acetyl-CoA molecules are produced from each glucose molecule, two cycles are required per glucose molecule.
- At the end of all cycles, the products are two GTP, six NADH, two FADH2, four CO2.
Combining the above reaction with the ones occuring in the course of glycolysis, we get the following overall glucose oxydation reaction before the respiratory chain:
(the above reactions are equilibrated if Pi represents the H2PO4- ion, ADP and GDP the ADP2- and GDP2- ions respectively, ATP and GTP the ATP3- and GTP3- ions respectively).
Considering the future conversion of GTP to ATP and the maximum 26 ATP produced by the 10 NADH and the 2 FADH2 in the electron transport chain, we see that each glucose molecule is able to produce a maximum of 30 ATP.
Although pyruvate dehydrogenase is not technically a part of the citric acid cycle, its regulation is included here.
Many of the enzymes in the TCA cycle are regulated by negative feedback from ATP when the energy charge of the cell is high. Such enzymes include the pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase. These enzymes, which regulate the first three steps of the TCA cycle, are inhibited by high concentrations of ATP. This regulation ensures that the TCA cycle will not oxidise excessive amounts of pyruvate and acetyl-CoA when ATP in the cell is plentiful. This type of negative regulation by ATP is by an allosteric mechanism.
Several enzymes are also negatively regulated when the level of reducing equivalents in a cell are high (high ratio of NADH/NAD+). This mechanism for regulation is due to substrate inhibition by NADH of the enzymes that use NAD+ as a substrate. This includes pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase.
Calcium is used as a regulator. It activates pyruvate dehydrogenase, isocitrate dehydrogenase and oxoglutarate dehydrogenase. This increases the reaction rate of many of the steps in the cycle, and therefore increases flux throughout the pathway.
Citrate is used for feedback inhibition, as it inhibits the phosphofructokinase(enzyme in glycolysis that makes Fructose 1,6-bisphosphate), a precursor of pyruvate. This prevents a constant high rate of flux when there is a build up of citrate and a decrease in substrate for the enzyme.
Most of the body's catabolic pathways converge on the TCA cycle, as the diagram shows. Reactions that form intermediates of the TCA cycle in order to replenish them (especially during the scarcity of the intermediates) are called anaplerotic reactions.
The citric acid cycle is the third step in carbohydrate catabolism (the breakdown of sugars). Glycolysis breaks glucose (a six-carbon-molecule) down into pyruvate (a three-carbon molecule). In eukaryotes, pyruvate moves into the mitochondria. It is converted into acetyl-CoA by decarboxylation and enters the citric acid cycle.
In protein catabolism, proteins are broken down by protease enzymes into their constituent amino acids. These amino acids are brought into the cells and can be a source of energy by being funnelled into the citric acid cycle.
In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In many tissues, especially heart tissue, fatty acids are broken down through a process known as beta oxidation which results in acetyl-CoA which can be used in the citric acid cycle. Sometimes beta oxidation can yield propionyl CoA which can result in further glucose production by gluconeogenesis in the liver.
The citric acid cycle is always followed by oxidative phosphorylation. This process extracts the energy (as electrons) from NADH and FADH2, oxidating them to NAD+ and FAD, respectively, so that the cycle can continue. The citric acid cycle itself does not use oxygen, but oxidative phosphorylation does.
The total energy gained from the complete breakdown of one molecule of glucose by glycolysis, the citric acid cycle and oxidative phosphorylation equals about 36 ATP molecules. The citric acid cycle is called an amphibolic pathway because it participates in both catabolism and anabolism.
MICROBIAL GENETICS AND MICROBIAL METABOLISM
Journey into the Cell: Mitochondria
Mitochondria: Architecture dictates function
A Brief History of Mitochondria
Mitochondria: Student Page http://www.fi.edu/qa97/biology/cells/cell9.html
The Virtual Cell Website http://personal.tmlp.com/Jimr57/textbook/chapter3/chapter3.htm
From Wikipedia, the free encyclopedia
6.5 (secondary phosphate)
Adenosine 5'-triphosphate (ATP) is a multifunctional nucleotide that is most important as a "molecular currency" of intracellular energy transfer. In this role ATP transports chemical energy within cells for metabolism. It is produced as an energy source during the processes of photosynthesis and cellular respiration and consumed by many enzymes and a multitude of cellular processes including biosynthetic reactions, motility and cell division. ATP is also incorporated into nucleic acids by polymerases in the processes of DNA replication and transcription. In signal transduction pathways, ATP is used as a substrate by kinases that phosphorylate proteins and lipids, as well as by adenylate cyclase, which uses ATP to produce the second messenger molecule cyclic AMP.
The structure of this molecule consists of a purine base (adenine) attached to the 1' carbon atom of a pentose (ribose). Three phosphate groups are attached at the 5' carbon atom of the pentose sugar. When ATP is used in DNA synthesis, the ribose sugar is first converted to deoxyribose by ribonucleotide reductase. ATP was discovered in 1929 by Karl Lohmann, and was proposed to be the main energy-transfer molecule in the cell by Fritz Albert Lipmann in 1941. The Citric Acid Cycle
Krebs identified the citric acid cycle in 1937 by noting that small quantities of organic acids such as succinate, malate or citrate stimulated oxygen uptake by minced pigeon breast muscle. He realised that the amount of additional oxygen consumed was greater than that required for the complete oxidation of the added material, and deduced that the acids must act catalytically.
Most of the oxygen was consumed by the endogenous substrates that were already present in the mince. Using inhibitors (such as malonate, which blocks succinate oxidation) he showed that the supplementary organic acids participated in the oxidative processes, but they were normally regenerated by the tissue metabolism. Although several key intermediates and cofactors remained to be discovered, there was only one plausible sequence for the known participants and the cycle was established.
"Nature" rejected Krebs' paper as insufficiently important, and he was obliged to publish his results in "Experientia" instead.
The development of faster centrifuges in the late 1940's permitted the isolation of subcellular organelles such as nuclei and mitochondria. It was realised that the Krebs cycle took place within this particulate fraction, and that the enzyme activies were "latent" until the surrounding membranes were disrupted by mechanical forces or osmotic shock.
The detailed chemical structures have very limited medical significance, but you will find it very much easier to make sense of the other material in this course if you take the trouble to learn them! It may be helpful to follow one particular atom in acetyl CoA all the way round the cycle until it is lost as carbon dioxide, and the coloured boxes are intended to assist this process.
The Krebs cycle enzymes are soluble proteins located in the mitochondrial matrix space, except for succinate dehydrogenase, which is an integral membrane protein that is firmly attached to the inner surface of the inner mitochondrial membrane, where it communicates directly with components in the respiratory chain. Succinate dehydrogenase uses FAD as a prosthetic group, but the other three oxidation steps use NAD as their coenzyme. Most of the energy is captured when the reduced coenzymes are re-oxidised by the respiratory chain in the mitochondrial inner membrane, but there is a single "substrate level" phosphorylation catalysed by succinate thiokinase, which rather unusually uses GDP as its phosphate acceptor. We do not properly understand the reason for this. Despite the claims in some text books, the GTP may not communicate freely with other nucleotide pools.
The four dehydrogenase reactions differ considerably in their energy yield. Succinate has the lowest redox potential (the worst reducing agent) so its oxidation yields relatively little energy. Reoxidation of the FAD2H cofactor for succinate dehydrogenase by the respiratory chain will only support the formation of 1.5 ATP / mole. Malate is a better reducing agent, and will generate 2.5 ATP / mole when the NADH from malate dehydrogenase is re-cycled to NAD by the respiratory chain. Oxoglutatarate and especially isocitrate are even better reducing agents. They are restricted to 2.5 ATP / mole when they use NADH as their initial hydrogen carrier, but cells find it necessary to regulate these highly favourable reactions as described below.
Each turn of the cycle achieves the complete oxidation of one molecule of acetyl CoA to form 2 molecules of carbon dioxide. The process also yields 3 molecules of NADH, 1 of FAD2H and one molecule of high-energy phosphate in the form of GTP. Reoxidation of all the reduced coenzymes by the respiratory chain yields a further 9 ATP, so the total energy captured by the cycle is 10 ATP equivalents per mole of acetyl CoA. This represents an overall energy conversion efficiency of about 62 % [free energy change for the complete oxidation of acetic acid = 805 kJ/mole, free energy of hydrolysis of ATP = 50 kJ/mole under typical intracellular conditions].
intermediates are present at very different concentrations. Citrate,
oxoglutarate, succinate and malate are major metabolites, and are typically
present at concentrations approaching
Krebs cycle enzymes redistribute material between the various pools, but they do not change the total amount of substrate in circulation. Acetyl CoA, for example, is oxidised via the cycle, but its incorporation into citrate merely "robs Peter to pay Paul" and it does not change the total quantity of cycle intermediates. The availability of the four-, five- and six-carbon organic acids affects the activity of the cycle in vivo, just as Krebs observed in his original in vitro experiments in 1937.
Krebs cycle intermediate concentrations depend on the activity of ancillary enzymes, which add material to the cycle from amino acid or carbohydrate sources, or alternatively remove intermediates for use in biosynthetic reactions. An important anaplerotic (or "filling up") reaction is catalysed by pyruvate carboxylase, which forms oxaloacetate from pyruvate within the mitochondria and is powerfully activated by acetyl CoA. The degradation of most amino acids also adds considerably to the total pool of cycle intermediates. The most important emptying processes are the removal of malate and related compounds for the synthesis of carbohydrates, and the export of citrate from the mitochondria for the biosynthesis of fat.
Another major factor affecting Krebs cycle activity is the availability of cofactors such as acetyl CoA, CoASH, NAD, FAD and GDP. Since the reactions form a closed loop, all five requirements must be simultaneously satisfied before the complete cycle can proceed. There is competition between the various oxidative pathways for access to the mitochondrial respiratory chain, so that it is possible, for example, for fatty acid oxidation to proceed at high speed (leading to the formation of ketone bodies) while the Krebs cycle is almost at a standstill.
Two allosteric enzymes help to control the distribution of metabolites and the overall cycle flux. The NAD-linked isocitrate dehydrogenase is inhibited by ATP, NADH & NADPH, and activated by ADP & calcium ions. Oxoglutarate dehydrogenase is also activated by calcium. Segments of the cycle may still be active even when the complete cycle cannot take place. This allows the cycle to perform its "clearing house" functions independently of respiratory activity and oxidative phosphorylation.
Energy stores and inter-conversions in humans
Notes: (1) These figures are for a 70kg male. The average daily energy intake is about 12 MJ per day for males, 9.2 MJ for females, so the total stores would last about 40 days, providing water was available and blood glucose could be maintained through gluconeogenesis. In practice food withdrawal may not be complete, and reduced physical activity lowers fasting energy requirements. Human beings have evolved to withstand a bad winter in a primitive hunter-gatherer society.
(2) There is no net synthesis of amino acids under physiological conditions, but in the case of the non-essential amino acids it may be possible to use transamination "to rob Peter to pay Paul". aconitase (mitochondrial) citrate synthase fumarase (mitochondrial) isocitrate dehydrogenase 3 (NAD, mitochondrial) malate dehydrogenase (mitochondrial) methylmalonyl CoA mutase methylmalonyl CoA racemase oxoglutarate dehydrogenase propionyl CoA carboxylase pyruvate dehydrogenase succinate dehydrogenase succinate thiokinase
ATP consists of adenosine - itself composed of an adenine ring and a ribose sugar - and three phosphate groups (triphosphate). The phosphoryl groups, starting with the group closest to the ribose, are referred to as the alpha (α), beta (β), and gamma (γ) phosphates. ATP is highly soluble in water and is quite stable in solutions between pH 6.8-7.4, but is rapidly hydrolysed at extreme pH, consequently ATP is best stored as an anhydrous salt.
The system of ATP and water under standard conditions and concentrations is extremely rich in chemical energy; the bond between the second and third phosphate groups is loosely said to be particularly high in energy. Strictly speaking, the bond itself is not high in energy (like all chemical bonds it requires energy to break), but energy is produced when the bond is broken and water is allowed to react with the two products. Thus, energy is produced from the new bonds formed between ADP and water, and between phosphate and water. The net change in enthalpy at Standard Temperature and Pressure of the decomposition of ATP into hydrated ADP and hydrated inorganic phosphate is -20.5 kJ / mole, with a change in free energy of 3.4 kJ/mole. This large release in energy makes the decomposition of ATP in water extremely exergonic, and hence useful as a means for chemically storing energy.
ATP has multiple ionizable groups with different acid dissociation constants. In neutral solution, ATP is ionized and exists mostly as ATP4−, with a small proportion of ATP3−. As ATP has several negatively-charged groups in neutral solution, it can chelate metals with very high affinity. The binding constant for various metal ions are (given as per mole) as Mg2+ (9 554), Na+ (13), Ca2+ (3 722), K+ (8), Sr2+ (1 381) and Li+ (25). Due to the strength of these interactions, ATP exists in the cell mostly in a complex with Mg2+.
Space-filling model of ATP
Ball-and-stick model of ATP
ATP can be produced by redox reactions using simple and complex sugars (carbohydrates) or lipids as an energy source. For ATP to be synthesized from complex fuels, they first need to be broken down into their basic components. Carbohydrates are hydrolysed into simple sugars, such as glucose and fructose. Fats (triglycerides) are metabolised to give fatty acids and glycerol.
The overall process of oxidizing glucose to carbon dioxide is known as cellular respiration and can produce up to 30 molecules of ATP from a single molecule of glucose. ATP can be produced by a number of distinct cellular processes; the three main pathways used to generate energy in eukaryotic organisms are glycolysis, the citric acid cycle/oxidative phosphorylation, and beta-oxidation. The majority of this ATP production by a non-photosynthetic aerobic eukaryote takes place in the mitochondria, which can make up nearly 25% of the total volume of a typical cell.
Main article: glycolysis
In glycolysis, glucose and glycerol are metabolized to pyruvate via the glycolytic pathway. In most organisms this process occurs in the cytosol, but in some protozoa such as the kinetoplastids, this is carried out in a specialized organelle called the glycosome. Glycolysis generates a net two molecules of ATP through substrate phosphorylation catalyzed by two enzymes: PGK and pyruvate kinase. Two molecules of NADH are also produced, which can be oxidized via the electron transport chain and result in the generation of additional ATP by ATP synthase. The pyruvate generated as an end-product of glycolysis is a substrate for the Krebs Cycle.
 Citric acid cycle
In the mitochondrion, pyruvate is oxidized by the pyruvate dehydrogenase complex to acetyl CoA, which is fully oxidized to carbon dioxide by the citric acid cycle (also known as the Krebs Cycle). Every "turn" of the citric acid cycle produces two molecules of carbon dioxide, one molecule of the ATP equivalent guanosine triphosphate (GTP) through substrate-level phosphorylation catalyzed by succinyl CoA synthetase, three molecules of the reduced coenzyme NADH, and one molecule of the reduced coenzyme FADH2. Both of these latter molecules are recycled to their oxidized states (NAD+ and FAD, respectively) via the electron transport chain, which generates additional ATP by oxidative phosphorylation. The oxidation of an NADH molecule results in the synthesis of about 3 ATP molecules, and the oxidation of one FADH2 yields about 2 ATP molecules. The majority of cellular ATP is generated by this process. Although the citric acid cycle itself does not involve molecular oxygen, it is an obligately aerobic process because O2 is needed to recycle the reduced NADH and FADH2 to their oxidized states. In the absence of oxygen the citric acid cycle will cease to function due to the lack of available NAD+ and FAD.
The generation of ATP by the mitochondrion from cytosolic NADH relies on the malate-aspartate shuttle (and to a lesser extent, the glycerol-phosphate shuttle) because the inner mitochondrial membrane is impermeable to NADH and NAD+. Instead of transferring the generated NADH, a malate dehydrogenase enzyme converts oxaloacetate to malate, which is translocated to the mitochondrial matrix. Another malate dehydrogenase-catalyzed reaction occurs in the opposite direction, producing oxaloacetate and NADH from the newly transported malate and the mitochondrion's interior store of NAD+. A transaminase converts the oxaloacetate to aspartate for transport back across the membrane and into the intermembrane space.
In oxidative phosphorylation, the passage of electrons from NADH and FADH2 through the electron transport chain powers the pumping of protons out of the mitrochondrial matrix and into the intermembrane space. This creates a proton motive force that is the net effect of a pH gradient and an electric potential gradient across the inner mitochondrial membrane. Flow of protons down this potential gradient - that is, from the intermembrane space to the matrix - provides the driving force for ATP synthesis by the protein complex ATP synthase. This enzyme contains a rotor subunit that physically rotates relative to the static portions of the protein during ATP synthesis.
Most of the ATP synthesized in the mitochondria will be used for cellular processes in the cytosol; thus it must be exported from its site of synthesis in the mitochondrial matrix. The inner membrane contains antiporters that are integral membrane proteins used to exchange newly-synthesized ATP in the matrix for ADP in the intermembrane space.
Main article: beta-oxidation
Fatty acids can also be broken down to acetyl-CoA by beta-oxidation. Each turn of this cycle reduces the length of the acyl chain by two carbon atoms and produces one NADH and one FADH2 molecule, which are used to generate ATP by oxidative phosphorylation. Because NADH and FADH2 are energy-rich molecules, dozens of ATP molecules can be generated by the beta-oxidation of a single long acyl chain. The high energy yield of this process explains why fat is the best source of dietary calories.
Main article: anaerobic respiration
Anaerobic respiration or fermentation entails the generation of energy via the process of oxidation in the absence of O2 as an electron acceptor. In most eukaryotes, glucose is used as both an energy store and an electron donor. The equation for the oxidation of glucose to lactic acid is:
C6H12O6 ---> 2CH3CH(OH)COOH + 2 ATP
In prokaryotes, multiple electron acceptors can be used in anerobic respiration. These include nitrate, sulfate or carbon dioxide. These processes lead to the ecologically-important processes of denitrification, sulfate reduction and acetogenesis, respectively.
 ATP replenishment by nucleoside diphosphate kinases
ATP can also be synthesized through several so-called "replenishment" reactions catalyzed by the enzyme families of nucleoside diphosphate kinases (NDKs), which use other nucleoside triphosphates as a high-energy phosphate donor, and the ATP:guanido-phosphotransferase family, which uses creatine.
In plants, ATP is synthesized in thylakoid membrane of the chloroplast during the light-dependent reactions of photosynthesis in a process called photophosphorylation. Here, light energy is used to pump protons across the chloroplast membrane. This produces a proton-motive force and this drives the ATP synthase, exactly as in oxidative phosphorylation. Some of the ATP produced in the chloroplasts is consumed in the Calvin cycle, which produces triose sugars.
 ATP recycling
The total quantity of ATP in the human body is about 0.1 mole. The majority of ATP is not usually synthesised de novo, but is generated from ADP by the aforementioned processes. Thus, at any given time, the total amount of ATP + ADP remains fairly constant.
energy used by human cells requires the hydrolysis
of 100 to 150 moles of ATP daily which is around 50 to
ATP production in an aerobic eukaryotic cell is tightly regulated by allosteric mechanisms, by feedback effects, and by the substrate concentration dependence of individual enzymes within the glycolysis and oxidative phosphorylation pathways. Key control points occur in enzymatic reactions that are so energetically favorable that they are effectively irreversible under physiological conditions.
In glycolysis, hexokinase is directly inhibited by its product, glucose-6-phosphate, and pyruvate kinase is inhibited by ATP itself. The main control point for the glycolytic pathway is phosphofructokinase (PFK), which is allosterically inhibited by high concentrations of ATP and activated by high concentrations of AMP. The inhibition of PFK by ATP is unusual, since ATP is also a substrate in the reaction catalyzed by PFK; the biologically active form of the enzyme is a tetramer that exists in two possible conformations, only one of which binds the second substrate fructose-6-phosphate (F6P). The protein has two binding sites for ATP - the active site is accessible in either protein conformation, but ATP binding to the inhibitor site stabilizes the conformation that binds F6P poorly. A number of other small molecules can compensate for the ATP-induced shift in equilibrium conformation and reactivate PFK, including cyclic AMP, ammonium ions, inorganic phosphate, and fructose 1,6 and 2,6 biphosphate.
The citric acid cycle is regulated mainly by the availability of key substrates, particularly the ratio of NAD+ to NADH and the concentrations of calcium, inorganic phosphate, ATP, ADP, and AMP. Citrate - the molecule that gives its name to the cycle - is a feedback inhibitor of citrate synthase and also inhibits PFK, providing a direct link between the regulation of the citric acid cycle and glycolysis.
In oxidative phosphorylation, the key control point is the reaction catalyzed by cytochrome c oxidase, which is regulated by the availability of its substrate, the reduced form of cytochrome c. The amount of reduced cytochrome c available is directly related to the amounts of other substrates:
which directly implies this equation:
Thus, a high ratio of [NADH] to [NAD+] or a low ratio of [ADP][Pi] to [ATP] imply a high amount of reduced cytochrome c and a high level of cytochrome c oxidase activity. An additional level of regulation is introduced by the transport rates of ATP and NADH between the mitochondrial matrix and the cytoplasm.
ATP is generated in the cell by energy-releasing processes and is broken down by energy-consuming processes, in this way ATP transfers energy between spatially-separate metabolic reactions. ATP is the main energy source for the majority of cellular functions. This includes the synthesis of macromolecules, including DNA, RNA, and proteins. ATP also plays a critical role in the transport of macromolecules across cell membranes, e.g. exocytosis and endocytosis.
ATP is critically involved in maintaining cell structure by facilitating assembly and disassembly of elements of the cytoskeleton. In a related process, ATP is required for the shortening of actin and myosin filament crossbridges required for muscle contraction. This latter process is one of the main energy requirements of animals and is essential for locomotion and respiration.
humans, this signaling role is important in both the central and peripheral
nervous system. Activity-dependent release of ATP from synapses, axons and glia
activates purinergic membrane receptors known as P2. The P2Y receptors are metabotropic, i.e. G protein-coupled and modulate mainly
intracellular calcium and sometimes cyclic AMP levels. Fifteen members of the
P2Y family have been reported (P2Y1–P2Y15), although some are only related
through weak homology and several (P2Y5, P2Y7, P2Y9, P2Y10) do not function as
receptors that raise cytosolic calcium. The P2X ionotropic receptor
subgroup comprises seven members (P2X1–P2X7) which are ligand-gated Ca2+-permeable
ion channels that open when bound to an extracellular purine nucleotide. In
contrast to P2 receptors (agonist order ATP > ADP > AMP >
ATP is critical in signal transduction processes. It is used by kinases as the source of phosphate groups in their phosphate transfer reactions. Kinase activity on substrates such as proteins or membrane lipids are a common form of signal transduction. Phosphorylation of a protein by a kinase can activate or inhibit the target's activity, these proteins may themselves be kinases, and form part of a signal transduction cascade such as the mitogen-activated protein kinase cascade.
ATP is also used by adenylate cyclase and is transformed to the second messenger molecule cyclic AMP, which is involved in triggering calcium signals by the release of calcium from intracellular stores. This form of signal transduction is particularly important in brain function, although it is involved in the regulation of a multitude of other cellular processes.
In all known organisms, the deoxyribonucleotides that make up DNA are synthesized by the action of ribonucleotide reductase (RNR) enzymes on their corresponding ribonucleotides. This enzyme reduces the 2' hydroxyl group on the ribose sugar to deoxyribose, forming a deoxyribonucleotide (denoted dATP). All ribonucleotide reductase enzymes use a common sulfhydryl radical mechanism reliant on reactive cysteine residues that oxidize to form disulfide bonds in the course of the reaction. RNR enzymes are recycled by reaction with thioredoxin or glutaredoxin.
The regulation of RNR and related enzymes maintains a balance of dNTPs relative to each other and relative to NTPs in the cell. Very low dNTP concentration inhibits DNA synthesis and DNA repair and is lethal to the cell, while an abnormal ratio of dNTPs is mutagenic due to the increased likelihood of misincorporating a dNTP during DNA synthesis. Regulation of or differential specificity of RNR has been proposed as a mechanism for alterations in the relative sizes of intracellular dNTP pools under cellular stress such as hypoxia.
Some proteins that bind ATP do so in a characteristic protein fold known as the Rossmann fold, which is a general nucleotide-binding structural domain that can also bind the cofactor NAD. The most common ATP-binding proteins, known as kinases, share a small number of common folds; the protein kinases, the largest kinase superfamily, all share common structural features specialized for ATP binding and phosphate transfer.
ATP in complexes with proteins generally requires the presence of a divalent cation, almost always magnesium, which binds to the ATP phosphate groups. The presence of magnesium greatly decreases the dissociation constant of ATP from its protein binding partner without affecting the ability of the enzyme to catalyze its reaction once the ATP has bound. The presence of magnesium ions can serve as a mechanism for kinase regulation.
Biochemistry laboratories often use in vitro studies to explore ATP-dependent molecular processes. Enzyme inhibitors of ATP-dependent enzymes such as kinases are needed to experimentally examine the binding sites and transition states involved in ATP-dependent reactions. ATP analogs are also used in X-ray crystallography to determine a protein structure in complex with ATP, often together with other substrates. Most useful ATP analogs cannot be hydrolyzed as ATP would be; instead they trap the enzyme in a structure closely related to the ATP-bound state. Adenosine 5'-(gamma-thiotriphosphate) is an extremely common ATP analog in which one of the gamma-phosphate oxygens is replaced by a sulfur atom; this molecule is hydrolyzed at a dramatically slower rate than ATP itself and functions as an inhibitor of ATP-dependent processes. In crystallographic studies, hydrolysis transition states are modeled by the bound vanadate ion. However, caution is warranted in interpreting the results of experiments using ATP analogs, since some enzymes can hydrolyze them at appreciable rates at high concentration.
ATP and Other Nucleoside Triphosphates or: Bonds Rich in Energy
ATP is regarded as a universal source of energy occuring in all cell types. It is produced mainly during the oxidation of energy-rich (reduced) compounds processed in the respiratory chain and in photosynthesis.
ATP is needed
as a source of energy for biochemical syntheses
for transport processes (active transport) and
for mechanical work like movements (ciliar movements, plasma currents etc.)
ATP occurs usually as a magnesium or a manganese salt. For its hydrolysis, magnesium ions are necessary. Whenever it is spoken of ATP-degradation, it is always the hydrolysis of the terminal phosphate group(s) that is meant. The reactions are reversible:
ATP + H2O < > ADP + H3PO4 (= Pi)
ATP + H2O < > AMP + pyrophosphate (= PP)
ADP + H2O < > AMP + Pi
The phosphates are linked anhydrously, the innermost phosphate residue and the sugar residue are linked by an ester bond. Hydrolysis depends on the pH. The delta G° is -7.3 kcal/mol (ca -30.6 kJ/mol) at pH 7, i.e. at almost physiological conditions. It increases with a rising pH and is -10 kcal/mol (ca -42 kJ/mol) at pH
Since the delta G° for the breakdown of a pyrophosphate and for that of one phosphate residue are roughly the same, ATP, ADP and AMP can rather easily be converted into each other:
ATP + AMP < > 2 ADP
The delta G of the ATP breakdown is not very high compared to other phosphorylated compounds. Under this aspect, the term 'energy-rich linkage' seems irritating, but it has gained acceptance in biochemical literature as its hydrolysis is easily performed (with the help of the respective enzyme) and the energy is actually useable. The reason for the rather easily broken down linkage is in the electron accumulation at the terminal phosphate residues. Identical charges (here they are negative) repel each other and are in this case neutralized by hydrolysis.
In many cases, the terminal phosphate residue that is cleaved off from the ATP is not given away into solution as a free inorganic phosphate, but is transferred onto another molecule that becomes consequently phosphorylated. This process works also the other way round: a phosphorylated compound with a delta G° > -8 kcal/mol (-34 kJ/mol) can transfer its phosphate residue to ADP that as a consequence becomes ATP.
UMP, UDP, UTP, CMP, CDP, CTP, GMP, GDP, GTP.
The triphosphate nucleosides of these compounds and those of ATP are components of RNA. They are integrated into the polymer by splitting off pyrophosphate ( = PP). The corresponding desoxyribose derivatives (dATP, dGTP, dCTP....) are necessary for DNA synthesis, where dTTP is used instead of dUTP. The terminal phosphate residues of all nucleoside di- and triphosphates are equally rich in energy. The energy set free by their hydrolysis is used for biosyntheses. They share the work equally: UTP is needed for the synthesis of polysaccharides, CTP for that of lipids and GTP for the synthesis of proteins and other molecules. These specificities are the results of the different selectivities of the enzymes, that control each of these metabolic pathways.
ATP AND BIOLOGICAL ENERGY
Table of Contents
The Nature of ATP | Back to Top
Adenosine triphosphate (ATP), the energy currency or coin of the cell pictured in Figfures 1 and 2, transfers energy from chemical bonds to endergonic (energy absorbing) reactions within the cell. Structurally, ATP consists of the adenine nucleotide (ribose sugar, adenine base, and phosphate group, PO4-2) plus two other phosphate groups.
Energy is stored in the covalent bonds between phosphates, with the greatest amount of energy (approximately 7 kcal/mole) in the bond between the second and third phosphate groups. This covalent bond is known as a pyrophosphate bond.
We can write the chemical reaction for the formation of ATP as:
a) in chemicalese: ADP + Pi + energy ----> ATP
b) in English: Adenosine diphosphate + inorganic Phosphate + energy produces Adenosine Triphosphate
The chemical formula for the expenditure/release of ATP energy can be written as:
a) in chemicalese: ATP ----> ADP + energy + Pi
b) in English Adenosine Triphosphate produces Adenosine diphosphate + energy + inorganic Phosphate
An analogy between ATP and rechargeable batteries is appropriate. The batteries are used, giving up their potential energy until it has all been converted into kinetic energy and heat/unusable energy. Recharged batteries (into which energy has been put) can be used only after the input of additional energy. Thus, ATP is the higher energy form (the recharged battery) while ADP is the lower energy form (the used battery). When the terminal (third) phosphate is cut loose, ATP becomes ADP (Adenosine diphosphate; di= two), and the stored energy is released for some biological process to utilize. The input of additional energy (plus a phosphate group) "recharges" ADP into ATP (as in my analogy the spent batteries are recharged by the input of additional energy).
How to Make ATP | Back to Top
Two processes convert ADP into ATP: 1) substrate-level phosphorylation; and 2) chemiosmosis. Substrate-level phosphorylation occurs in the cytoplasm when an enzyme attaches a third phosphate to the ADP (both ADP and the phosphates are the substrates on which the enzyme acts). This is illustrated in Figure 3.
Figure 3. Enzymes and the formation of NADH and ATP. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.
shown in Figure 4, involves more than the single enzyme of substrate-level
phosphorylation. Enzymes in chemiosmotic synthesis are arranged in an electron transport chain
that is embedded in a membrane. In eukaryotes this membrane is in either the chloroplast
According to the chemiosmosis hypothesis proposed by Peter Mitchell in
During chemiosmosis in eukaryotes, H+ ions are pumped across an organelle membrane by membrane "pump proteins" into a confined space (bounded by membranes) that contains numerous hydrogen ions. This is shown in Figure 4 and 5. The energy for the pumping comes from the coupled oxidation-reduction reactions in the electron transport chain. Electrons are passed from one membrane-bound enzyme to another, losing some energy with each tansfer (as per the second law of thermodynamics). This "lost" energy allows for the pumping of hydrogen ions against the concentration gradient (there are fewer hydrogen ions outside the confined space than there are inside the confined space). The confined hydrogens cannot pass back through the membrane. Their only exit is through the ATP synthesizing enzyme that is located in the confining membrane. As the hydrogen passes through the ATP synthesizing enzyme, energy from the enzyme is used to attach a third phosphate to ADP, converting it to ATP.
Usually the terminal phosphate is not simply removed, but instead is attached to another molecule. This process is known as phosphorylation.
W + ATP -----> W~P + ADP where W is any compound, for example:
glucose + ATP -----> glucose~P + ADP
Glucose can be converted into Glucose-6-phosphate by the addition of the phosphate group from ATP.
Learning Objectives Describe the components, organization, and functions of an electron transport system.
· ATP is composed of ribose, a five-carbon sugar, three phosphate groups, and adenine , a nitrogen-containing compound (also known as a nitrogenous base). What class of organic macromolecules is composed of monomers similar to ATP?
· ATP directly or indirectly delivers energy to almost all metabolic pathways. Explain the functioning of the ATP/ADP cycle.
· Adding a phosphate to a molecule is called phosphorylation. What two methods do cells use to phosphorylate ADP into ATP?
ATP: The Perfect Energy Currency for the Cell
The major energy currency molecule of the cell, ATP, is evaluated in the context of creationism. This complex molecule is critical for all life from the simplest to the most complex. It is only one of millions of enormously intricate nanomachines that needs to have been designed in order for life to exist on earth. This motor is an excellent example of irreducible complexity because it is necessary in its entirety in order for even the simplest form of life to survive.
In order to function, every machine requires specific parts such as screws, springs, cams, gears, and pulleys. Likewise, all biological machines must have many well-engineered parts to work. Examples include units called organs such as the liver, kidney, and heart. These complex life units are made from still smaller parts called cells which in turn are constructed from yet smaller machines known as organelles. Cell organelles include mitochondria, Golgi complexes, microtubules, and centrioles. Even below this level are other parts so small that they are formally classified as macromolecules (large molecules).
Fig. 1. Views of ATP and related structures.
A critically important macromolecule—arguably “second in importance only to DNA”—is ATP. ATP is a complex nanomachine that serves as the primary energy currency of the cell (Trefil, 1992, p.93). A nanomachine is a complex precision microscopic-sized machine that fits the standard definition of a machine. ATP is the “most widely distributed high-energy compound within the human body” (Ritter, 1996, p. 301). This ubiquitous molecule is “used to build complex molecules, contract muscles, generate electricity in nerves, and light fireflies. All fuel sources of Nature, all foodstuffs of living things, produce ATP, which in turn powers virtually every activity of the cell and organism. Imagine the metabolic confusion if this were not so: Each of the diverse foodstuffs would generate different energy currencies and each of the great variety of cellular functions would have to trade in its unique currency” (Kornberg, 1989, p. 62).
ATP is an abbreviation for adenosine triphosphate, a complex molecule that contains the nucleoside adenosine and a tail consisting of three phosphates. (See Figure 1 for a simple structural formula and a space filled model of ATP.) As far as known, all organisms from the simplest bacteria to humans use ATP as their primary energy currency. The energy level it carries is just the right amount for most biological reactions. Nutrients contain energy in low-energy covalent bonds which are not very useful to do most of kinds of work in the cells.
These low energy bonds must be translated to high energy bonds, and this is a role of ATP. A steady supply of ATP is so critical that a poison which attacks any of the proteins used in ATP production kills the organism in minutes. Certain cyanide compounds, for example, are poisonous because they bind to the copper atom in cytochrome oxidase. This binding blocks the electron transport system in the mitochondria where ATP manufacture occurs (Goodsell, 1996, p.74).
How ATP Transfers Energy
Energy is usually liberated from the ATP molecule to do work in the cell by a reaction that removes one of the phosphate-oxygen groups, leaving adenosine diphosphate (ADP). When the ATP converts to ADP, the ATP is said to be spent. Then the ADP is usually immediately recycled in the mitochondria where it is recharged and comes out again as ATP. In the words of Trefil (1992, p. 93) “hooking and unhooking that last phosphate [on ATP] is what keeps the whole world operating.”
The enormous amount of activity that occurs inside each of the approximately one hundred trillion human cells is shown by the fact that at any instant each cell contains about one billion ATP molecules. This amount is sufficient for that cell’s needs for only a few minutes and must be rapidly recycled. Given a hundred trillion cells in the average male, about 1023 or one sextillion ATP molecules normally exist in the body. For each ATP “the terminal phosphate is added and removed 3 times each minute” (Kornberg, 1989, p. 65).
The total human body content of ATP is only about
The Structure of ATP
ATP contains the purine base adenine and the sugar ribose which together form the nucleoside adenosine. The basic building blocks used to construct ATP are carbon, hydrogen, nitrogen, oxygen, and phosphorus which are assembled in a complex that contains the number of subatomic parts equivalent to over 500 hydrogen atoms. One phosphate ester bond and two phosphate anhydride bonds hold the three phosphates (PO4) and the ribose together. The construction also contains a b-N glycoside bond holding the ribose and the adenine together.
Fig. 2. The two-dimensional stick model of the adenosine phosphate family of molecules, showing the atom and bond arrangement.
Phosphates are well-known high-energy molecules, meaning that comparatively high levels of energy are released when the phosphate groups are removed. Actually, the high energy content is not the result of simply the phosphate bond but the total interaction of all the atoms within the ATP molecule.
Because the amount of energy released when the phosphate bond is broken is very close to that needed by the typical biological reaction, little energy is wasted. Generally, ATP is connected to another reaction—a process called coupling which means the two reactions occur at the same time and at the same place, usually utilizing the same enzyme complex. Release of phosphate from ATP is exothermic (a reaction that gives off heat) and the reaction it is connected to is endothermic (requires energy input in order to occur). The terminal phosphate group is then transferred by hydrolysis to another compound, a process called phosphorylation, producing ADP, phosphate (Pi) and energy.
The self-regulation system of ATP has been described as follows:
The high-energy bonds of ATP are actually rather unstable bonds. Because they are unstable, the energy of ATP is readily released when ATP is hydrolyzed in cellular reactions. Note that ATP is an energy-coupling agent and not a fuel. It is not a storehouse of energy set aside for some future need. Rather it is produced by one set of reactions and is almost immediately consumed by another. ATP is formed as it is needed, primarily by oxidative processes in the mitochondria. Oxygen is not consumed unless ADP and a phosphate molecule are available, and these do not become available until ATP is hydrolyzed by some energy-consuming process. Energy metabolism is therefore mostly self-regulating (Hickman, Roberts, and Larson, 1997, p.43). [Italics mine]
ATP is not excessively unstable, but it is designed so that its hydrolysis is slow in the absence of a catalyst. This insures that its stored energy is “released only in the presence of the appropriate enzyme” (McMurry and Castellion, 1996, p. 601).
The Function of ATP
The ATP is used for many cell functions including transport work moving substances across cell membranes. It is also used for mechanical work, supplying the energy needed for muscle contraction. It supplies energy not only to heart muscle (for blood circulation) and skeletal muscle (such as for gross body movement), but also to the chromosomes and flagella to enable them to carry out their many functions. A major role of ATP is in chemical work, supplying the needed energy to synthesize the multi-thousands of types of macromolecules that the cell needs to exist.
ATP is also used as an on-off switch both to control chemical reactions and to send messages. The shape of the protein chains that produce the building blocks and other structures used in life is mostly determined by weak chemical bonds that are easily broken and remade. These chains can shorten, lengthen, and change shape in response to the input or withdrawal of energy. The changes in the chains alter the shape of the protein and can also alter its function or cause it to become either active or inactive.
The ATP molecule can bond to one part of a protein molecule, causing another part of the same molecule to slide or move slightly which causes it to change its conformation, inactivating the molecule. Subsequent removal of ATP causes the protein to return to its original shape, and thus it is again functional. The cycle can be repeated until the molecule is recycled, effectively serving as an on and off switch (Hoagland and Dodson, 1995, p.104). Both adding a phosphorus (phosphorylation) and removing a phosphorus from a protein (dephosphorylation) can serve as either an on or an off switch.
How is ATP Produced?
ATP is manufactured as a result of several cell processes including fermentation, respiration and photosynthesis. Most commonly the cells use ADP as a precursor molecule and then add a phosphorus to it. In eukaryotes this can occur either in the soluble portion of the cytoplasm (cytosol) or in special energy-producing structures called mitochondria. Charging ADP to form ATP in the mitochondria is called chemiosmotic phosphorylation. This process occurs in specially constructed chambers located in the mitochondrion’s inner membranes.
Fig. 3. An outline of the ATP-synthase macromolecule showing its subunits and nanomachine traits. ATP-synthase converts ADP into ATP, a process called charging. Shown behind ATP-synthase is the membrane in which the ATP-synthase is mounted. For the ATP that is charged in the mitochondria, ATP-synthase is located in the inner membrane.
The mitochondrion itself functions to produce an electrical chemical gradient—somewhat like a battery—by accumulating hydrogen ions in the space between the inner and outer membrane. This energy comes from the estimated 10,000 enzyme chains in the membranous sacks on the mitochondrial walls. Most of the food energy for most organisms is produced by the electron transport chain. Cellular oxidation in the Krebs cycle causes an electron build-up that is used to push H+ ions outward across the inner mitochondrial membrane (Hickman et al., 1997, p. 71).
As the charge builds up, it provides an electrical potential that releases its energy by causing a flow of hydrogen ions across the inner membrane into the inner chamber. The energy causes an enzyme to be attached to ADP which catalyzes the addition of a third phosphorus to form ATP. Plants can also produce ATP in this manner in their mitochondria but plants can also produce ATP by using the energy of sunlight in chloroplasts as discussed later. In the case of eukaryotic animals the energy comes from food which is converted to pyruvate and then to acetyl coenzyme A (acetyl CoA). Acetyl CoA then enters the Krebs cycle which releases energy that results in the conversion of ADP back into ATP.
How does this potential difference serve to reattach the phosphates on ADP molecules? The more protons there are in an area, the more they repel each other. When the repulsion reaches a certain level, the hydrogens ions are forced out of a revolving-door-like structure mounted on the inner mitochondria membrane called ATP synthase complexes. This enzyme functions to reattach the phosphates to the ADP molecules, again forming ATP.
The ATP synthase revolving door resembles a molecular water wheel that harnesses the flow of hydrogen ions in order to build ATP molecules. Each revolution of the wheel requires the energy of about nine hydrogen ions returning into the mitochondrial inner chamber (Goodsell, 1996, p.74). Located on the ATP synthase are three active sites, each of which converts ADP to ATP with every turn of the wheel. Under maximum conditions, the ATP synthase wheel turns at a rate of up to 200 revolutions per second, producing 600 ATPs during that second.
ATP is used in conjunction with enzymes to cause certain molecules to bond together. The correct molecule first docks in the active site of the enzyme along with an ATP molecule. The enzyme then catalyzes the transfer of one of the ATP phosphates to the molecule, thereby transferring to that molecule the energy stored in the ATP molecule. Next a second molecule docks nearby at a second active site on the enzyme. The phosphate is then transferred to it, providing the energy needed to bond the two molecules now attached to the enzyme. Once they are bonded, the new molecule is released. This operation is similar to using a mechanical jig to properly position two pieces of metal which are then welded together. Once welded, they are released as a unit and the process then can begin again.
A Double Energy Packet
Although ATP contains the amount of energy necessary for most reactions, at times more energy is required. The solution is for ATP to release two phosphates instead of one, producing an adenosine monophosphate (AMP) plus a chain of two phosphates called a pyrophosphate. How adenosine monophosphate is built up into ATP again illustrates the precision and the complexity of the cell energy system. The enzymes used in glycolysis, the citric acid cycle, and the electron transport system, are all so precise that they will replace only a single phosphate. They cannot add two new phosphates to an AMP molecule to form ATP.
The solution is an intricate enzyme called adenylate kinase which transfers a single phosphate from an ATP to the AMP, producing two ADP molecules. The two ADP molecules can then enter the normal Krebs cycle designed to convert ADP into ATP. Adenylate kinase requires an atom of magnesium—and this is one of the reasons why sufficient dietary magnesium is important.
Adenylate kinase is a highly organized but compact enzyme with its active site located deep within the molecule. The deep active site is required because the reactions it catalyzes are sensitive to water. If water molecules lodged between the ATP and the AMP, then the phosphate might break ATP into ADP and a free phosphate instead of transferring a phosphate from ATP to AMP to form ADP.
To prevent this, adenylate kinase is designed so that the active site is at the end of a channel deep in the structure which closes around AMP and ATP, shielding the reaction from water. Many other enzymes that use ATP rely on this system to shelter their active site to prevent inappropriate reactions from occurring. This system ensures that the only waste that occurs is the normal wear, tear, repair, and replacement of the cell’s organelles.
Pyrophosphates and pyrophosphoric acid, both inorganic forms of phosphorus, must also be broken down so they can be recycled. This phosphate breakdown is accomplished by the inorganic enzyme pyrophosphatase which splits the pyrophosphate to form two free phosphates that can be used to charge ATP (Goodsell, 1996, p.79). This system is so amazingly efficient that it produces virtually no waste, which is astounding considering its enormously detailed structure. Goodsell (1996, p. 79) adds that “our energy-producing machinery is designed for the production of ATP: quickly, efficiently, and in large quantity.”
The main energy carrier the body uses is ATP, but other energized nucleotides are also utilized such as thymine, guanine, uracil, and cytosine for making RNA and DNA. The Krebs cycle charges only ADP, but the energy contained in ATP can be transferred to one of the other nucleosides by means of an enzyme called nucleoside diphosphate kinase. This enzyme transfers the phosphate from a nucleoside triphosphate, commonly ATP, to a nucleoside diphosphate such as guanosine diphosphate (GDP) to form guanosine triphosphate (GTP).
The nucleoside diphosphate kinase works by one of its six active sites binding nucleoside triphosphate and releasing the phosphate which is bonded to a histidine. Then the nucleoside triphosphate, which is now a diphosphate, is released, and a different nucleoside diphosphate binds to the same site—and as a result the phosphate that is bonded to the enzyme is transferred, forming a new triphosphate. Scores of other enzymes exist in order for ATP to transfer its energy to the various places where it is needed. Each enzyme must be specifically designed to carry out its unique function, and most of these enzymes are critical for health and life.
The body does contain some flexibility, and sometimes life is possible when one of these enzymes is defective—but the person is often handicapped. Also, back-up mechanisms sometimes exist so that the body can achieve the same goals through an alternative biochemical route. These few simple examples eloquently illustrate the concept of over-design built into the body. They also prove the enormous complexity of the body and its biochemistry.
The Message of the Molecule
Without ATP, life as we understand it could not exist. It is a perfectly-designed, intricate molecule that serves a critical role in providing the proper size energy packet for scores of thousands of classes of reactions that occur in all forms of life. Even viruses rely on an ATP molecule identical to that used in humans. The ATP energy system is quick, highly efficient, produces a rapid turnover of ATP, and can rapidly respond to energy demand changes (Goodsell, 1996, p.79).
Furthermore, the ATP molecule is so enormously intricate that we are just now beginning to understand how it works. Each ATP molecule is over 500 atomic mass units (500 AMUs). In manufacturing terms, the ATP molecule is a machine with a level of organization on the order of a research microscope or a standard television (Darnell, Lodish, and Baltimore, 1996).
Among the questions evolutionists must answer include the following, “How did life exist before ATP?” “How could life survive without ATP since no form of life we know of today can do that?” and “How could ATP evolve and where are the many transitional forms required to evolve the complex ATP molecule?” No feasible candidates exist and none can exist because only a perfect ATP molecule can properly carry out its role in the cell.
In addition, a potential ATP candidate molecule would not be selected for by evolution until it was functional and life could not exist without ATP or a similar molecule that would have the same function. ATP is an example of a molecule that displays irreducible complexity which cannot be simplified and still function (Behe, 1996). ATP could have been created only as a unit to function immediately in life and the same is true of the other intricate energy molecules used in life such as GTP.
Although other energy molecules can be used for certain cell functions, none can even come close to satisfactorily replacing all the many functions of ATP. Over 100,000 other detailed molecules like ATP have also been designed to enable humans to live, and all the same problems related to their origin exist for them all. Many macromolecules that have greater detail than ATP exist, as do a few that are less highly organized, and in order for life to exist all of them must work together as a unit.
The Contrast between Prokaryotic and Eukaryotic ATP Production An enormous gap exists between prokaryote (bacteria and cyanobacteria) cells and eukaryote (protists, plants and animals) type of cells:
...prokaryotes and eukaryotes are profoundly different from each other and clearly represent a marked dichotomy in the evolution of life. . . The organizational complexity of the eukaryotes is so much greater than that of the prokaryotes that it is difficult to visualize how a eukaryote could have arisen from any known prokaryote (Hickman et al., 1997, p. 39).
Some of the differences are that prokaryotes lack organelles, a cytoskeleton, and most of the other structures present in eukaryotic cells. Consequently, the functions of most organelles and other ultrastructure cell parts must be performed in bacteria by the cell membrane and its infoldings called mesosomes.
The Four Major Methods of Producing ATP
A crucial difference between prokaryotes and eukaryotes is the means they use to produce ATP. All life produces ATP by three basic chemical methods only: oxidative phosphorylation, photophosphorylation, and substrate-level phosphorylation (Lim, 1998, p. 149). In prokaryotes ATP is produced both in the cell wall and in the cytosol by glycolysis. In eukaryotes most ATP is produced in chloroplasts (for plants), or in mitochondria (for both plants and animals). No means of producing ATP exists that is intermediate between these four basic methods and no transitional forms have ever been found that bridge the gap between these four different forms of ATP production. The machinery required to manufacture ATP is so intricate that viruses are not able to make their own ATP. They require cells to manufacture it and viruses have no source of energy apart from cells.
In prokaryotes the cell membrane takes care of not only the cell’s energy-conversion needs, but also nutrient processing, synthesizing of structural macromolecules, and secretion of the many enzymes needed for life (Talaro and Talaro, 1993, p. 77). The cell membrane must for this reason be compared with the entire eukaryote cell ultrastructure which performs these many functions. No simple means of producing ATP is known and prokaryotes are not by any means simple. They contain over 5,000 different kinds of molecules and can use sunlight, organic compounds such as carbohydrates, and inorganic compounds as sources of energy to manufacture ATP.
Another example of the cell membrane in prokaryotes assuming a function of the eukaryotic cell ultrastructure is as follows: Their DNA is physically attached to the bacterial cell membrane and DNA replication may be initiated by changes in the membrane. These membrane changes are in turn related to the bacterium’s growth. Further, the mesosome appears to guide the duplicated chromatin bodies into the two daughter cells during cell division (Talaro and Talaro, 1993).
In eukaryotes the mitochondria produce most of the cell’s ATP (anaerobic glycolysis also produces some) and in plants the chloroplasts can also service this function. The mitochondria produce ATP in their internal membrane system called the cristae. Since bacteria lack mitochondria, as well as an internal membrane system, they must produce ATP in their cell membrane which they do by two basic steps. The bacterial cell membrane contains a unique structure designed to produce ATP and no comparable structure has been found in any eukaryotic cell (Jensen, Wright, and Robinson, 1997).
In bacteria, the ATPase and the electron transport chain are located inside the cytoplasmic membrane between the hydrophobic tails of the phospholipid membrane inner and outer walls. Breakdown of sugar and other food causes the positively charged protons on the outside of the membrane to accumulate to a much higher concentration than they are on the membrane inside. This creates an excess positive charge on the outside of the membrane and a relatively negative charge on the inside.
The result of this charge difference
is a dissociation of H2O molecules into H+ and
In some bacteria such as Halobacterium this system is modified by use of bacteriorhodopsin, a protein similar to the sensory pigment rhodopsin used in the vertebrate retina (Lim, 1998, p. 166). Illumination causes the pigment to absorb light energy, temporarily changing rhodopsin from a trans to a cis form. The trans to cis conversion causes deprotonation and the transfer of protons across the plasma membrane to the periplasm.
The proton gradient that results is used to drive ATP synthesis by use of the ATPase complex. This modification allows bacteria to live in low oxygen but rich light regions. This anaerobic ATP manufacturing system, which is unique to prokaryotes, uses a chemical compound other than oxygen as a terminal electron acceptor (Lim, 1998, p. 168). The location of the ATP producing system is only one of many major contrasts that exist between bacterial cell membranes and mitochondria.
Chloroplasts are double membraned ATP-producing organelles found only in plants. Inside their outer membrane is a set of thin membranes organized into flattened sacs stacked up like coins called thylakoids (Greek thylac or sack, and oid meaning like). The disks contain chlorophyll pigments that absorb solar energy which is the ultimate source of energy for all the plant’s needs including manufacturing carbohydrates from carbon dioxide and water (Mader, 1996, p. 75). The chloroplasts first convert the solar energy into ATP stored energy, which is then used to manufacture storage carbohydrates which can be converted back into ATP when energy is needed.
The chloroplasts also possess an electron transport system for producing ATP. The electrons that enter the system are taken from water. During photosynthesis, carbon dioxide is reduced to a carbohydrate by energy obtained from ATP (Mader, 1996, p. 12). Photosynthesizing bacteria (cyanobacteria) use yet another system. Cyanobacteria do not manufacture chloroplasts but use chlorophyll bound to cytoplasmic thylakoids. Once again plausible transitional forms have never been found that can link this form of ATP production to the chloroplast photosynthesis system.
The two most common evolutionary theories of the origin of the mitochondria-chloroplast ATP production system are 1) endosymbiosis of mitochondria and chloroplasts from the bacterial membrane system and 2) the gradual evolution of the prokaryote cell membrane system of ATP production into the mitochondria and chloroplast systems. Believers in endosymbiosis teach that mitochondria were once free-living bacteria, and that “early in evolution ancestral eukaryotic cells simply ate their future partners” (Vogel, 1998, p. 1633). Both the gradual conversion and endosymbiosis theory require many transitional forms, each new one which must provide the animal with a competitive advantage compared with the unaltered animals.
The many contrasts between the prokaryotic and eukaryotic means of producing ATP, some of which were noted above, are strong evidence against the endosymbiosis theory. No intermediates to bridge these two systems has ever been found and arguments put forth in the theory’s support are all highly speculative. These and other problems have recently become more evident as a result of recent major challenges to the standard endosymbiosis theory. The standard theory has recently been under attack from several fronts, and some researchers are now arguing for a new theory:
Scientists pondering how the first complex cell came together
say the new idea could solve some nagging problems with the prevailing
theory... “[the new theory is]... elegantly argued,” says Michael Gray of
In this brief review we have examined only one cell macromolecule, ATP, and the intricate mechanisms which produce it. We have also looked at the detailed supporting mechanism which allows the ATP molecule to function. ATP is only one of hundreds of thousands of essential molecules, each one that has a story. As each of those stories is told, they will stand as a tribute to both the genius and the enormously complex design of the natural world. All the books in the largest library in the world may not be able to contain the information needed to understand and construct the estimated 100,000 complex macromolecule machines used in humans. Much progress has been made in understanding the structure and function of organic macromolecules and some of the simpler ones are now being manufactured by pharmaceutical firms.
Now that scientists understand how some of these highly organized molecules function and why they are required for life, their origin must be explained. We know only four basic methods of producing ATP: in bacterial cell walls, in the cytoplasm by photosynthesis, in chloroplasts, and in mitochondria. No transitional forms exist to bridge these four methods by evolution. According to the concept of irreducible complexity, these ATP producing machines must have been manufactured as functioning units and they could not have evolved by Darwinism mechanisms. Anything less than an entire ATP molecule will not function and a manufacturing plant which is less then complete cannot produce a functioning ATP. Some believe that the field of biochemistry which has achieved this understanding has already falsified the Darwinian world view (Behe, 1996).
A substance called adenosine triphosphate (ATP) links most cellular exergonic (def) and endergonic (def) chemical reactions. To obtain energy to do cellular work, organisms take energy-rich compounds such as glucose into the cell and enzymatically break them down to release their potential energy. Therefore, the organism needs a way to trap some of that released energy and store the energy in a form that can be utilized by the cell to do cellular work. Principally, energy is trapped and stored in the form of adenosine triphosphate (def) or ATP.
A tremendous amount of ATP is needed for normal cellular growth. For
example,a human at rest uses about
To trap energy released from exergonic catabolic chemical reactions (def), the cell uses some of that released energy to attach an inorganic phosphate group on to adenosine diphosphate (ADP) to make adenosine triphosphate (ATP). Thus, energy is trapped and stored in what are known as high-energy phosphate bonds. To obtain energy to do cellular work during endergonic anabolic chemical reactions (def), the organism enzymatically removes the third phosphate from ATP thus releasing the stored energy and forming ADP and inorganic phosphate once again (see Fig. 1).
Depending on the type of organism, cells transfer energy and generate ATP by photophosphorylation, by substrate-level phosphorylation, and/or by oxidative phosphorylation. (Phosphorylation (def) refers to the attachment of a phosphate group to a molecule.)
Oxidative phosphorylation in eukaryotes occurs exclusively in their mitochondria.
As previously discussed, mitochondria convert pyruvate into carbon dioxide and water via the Krebs cycle. This produces NADH and a little ATP. The NADH, which would otherwise accumulate until there was no NAD left, is re-oxidised by the oxidative phosphorylation respiratory chain to regenerate the NAD, and (as a fabulous bonus), generate loads of ATP too, because this oxidation is coupled to the production of a proton gradient across their inner membrane, and these protons flow down their gradient via FOF1-ATPase (ATP synthase), making ATP.
Mitochondria are believed to be the product of an endosymbiosis 2500 MYA. They probably originated from an intracellular proteobacterial parasite of proto-eukaryotic cells (something like Bdellovibrio). Chloroplasts derived from a similar symbiosis with cyanobacteria (closely related to Prochloron).
Since oxidative phosphorylation is much more efficient (30 vs. 2 ATP per glucose) than anaerobic respiration, and photosynthesis allows growth in the absence of exogenous carbon and reducing agents, what may have begun as a parasitic or predatory relationship between the Ur-eukaryote and its bacterial passengers, developed into a mutualism. The mitochondrial symbiosis were probably a one-off, but chloroplasts may not have been. Dinoflagellates even have secondary endosymbionts (like Russian matryoshka dolls): a chloroplast within an alga within another alga. There is a great deal of evidence for endosymbiosis these days (the theory was once considered very unlikely):
· Inner membrane = Remains of bacterial plasmalemma.
· Outer membrane = Remains of food vacuole.
· Cristae = Like mesosome infoldings.
· Circular DNA lacking histones in matrix = Remains of bacterial chromosome.
· Small ribosomes = Like bacterial 70S ribosomes (eukaryotes have 80S).
The symbiosis has gone far beyond a simple ingestion. The human mtDNA (mitochondrial DNA) genome contains just 37 genes. These are mostly tRNAs, with some of the proteins of oxidative phosphorylation:
· 7/27 of Complex I
· 0/4 of Complex II
· 1/9 of Complex III
· 3/13 of Complex IV
· 2/12 of Complex V
The rest is now nuclear encoded and imported via the TOM/TIM transport system, showing that over evolutionary time, the genes of the mitochondria have either been lost, or (when essential) transferred to the nuclear genome.
The job of mitochondria is to convert pyruvate to ATP and carbon dioxide. This is achieved by the interaction of NADH, and one Krebs cycle intermediate (succinate) with the inner mitochondrial membrane. This membrane contains five huge protein complexes, which serve to remove electrons from NADH, regenerating NAD, and in-so-doing, to generate a proton gradient across the membrane than may be used to drive ATP synthesis.
The five complexes are named I, II, III, IV and V. We shall discuss them in turn:
Electrons from the oxidation of NADH pass through flavin,
Complex I is NADH dehydrogenase. It removes two electron from NADH, and transfers them to ubiquinone in the mitochondrial membrane. As the two electrons pass through various flavin (FMN), iron-sulfur (FeS) and quinone (UQ) centres, four protons are pumped across complex I into the inter-membrane space (per NADH). When the electrons are deposited onto UQ (ubiquinone), the UQ takes up a further two protons from the matrix side, to form ubiquinol (UQH2) (these are excluded from the pump-count for this complex). It produces 1 UQH2, per NADH oxidised. The ultimate source of the NADH and the electrons is the oxidation of ketoglutarate, malate etc in the Krebs cycle.
The ubiquinol formed feeds into a UQ 'pool' inside the membrane, and diffuses to complex III.
Succinate is oxidised to generate fumarate, and UQH2 from UQ.
Complex II is also called succinate dehydrogenase, and is the only membrane bound enzyme of the Krebs cycle. The dehydrogenation of succinate has too small a ∆G for any H+ pumping, so this complex only generates 1 UQH2 per succinate oxidised, and pumps no protons. It gets its electrons from the oxidation of succinate only, and feeds them via flavin (FAD) and an iron-sulfur cluster into the UQH2 pool.
Ubiquinone and ubiquinol
Ubiquinone (UQ) ferries electrons from complexes I and II to complex III. Note the long hydrophobic chain: UQ/UQH2 can migrate actually dissolved within the membrane.
Partial reduction of UQ generates ubisemiquinone radicals (UQH·), which are very dangerous and must be rapidly reduced to UQH2.
Complexes I and II both feed into a pool of ubiquinol (UQH2) actually inside the inner mitochondrial membrane (dissolved in the fatty acid tails).
The reason semiubiquinone is dangerous is that is can generate superoxide radicals, which are hugely oxidising free-radicals.
UQH· + O2 → UQ + H+ + O2−·
Superoxide will dismutate to hydrogen peroxide.
2O2−· + 2H+ → O2 + H2O2
Hydrogen peroxide will undergo Fenton reaction with haem iron to produce hydroxyl radicals which are lethally destructive.
Fe2+ + H2O2 → Fe3+ + OH− + OH·
Mitochondria therefore contain superoxide dismutase and glutathione (GSH) peroxidase to cope with these agents of oxidative stress.
2GSH + H2O2 → GSSG + 2H2O
This is also called cytochrome reductase (or oxidoreductase). It pumps 4 H+ per UQH2 (including the two attached by complex I or II to UQ), and produces 2 cyt-cRED (reduced cytochrome-c) per UQH2 oxidised. The iron in the haem groups of b and c cytochromes goes from Fe3+ to Fe2+. The complex manages to pump 4 protons by running a nasty bit of biochemistry called the Q-cycle, which delivers the two electrons from one UQH2 to two cyt-c molecules, which only carry one electron each.
'Q-cycle' is a preposterously complicated way of transferring electrons from
the two-electron-carrying UQH2 to the single-electron carrying
cytochrome-c (cyt-c). A UQH2 gives up its protons to the IMS. One of
its electrons is carried through
One electron from UQH2 is used to reduce cyt-c, the other is used to half-reduce a UQ in the membrane to semiquinone. This is accompanied by the release of two protons per UQH2 into the IMS. One electron from a second UQH2 is used to reduce cyt-c, and the other is used to regenerate a UQH2 in the membrane from the semiquinone radical produced earlier, with uptake of protons from the matrix. This is again accompanied by the release of two protons per UQH2 into the IMS. In upshot, only one net UQH2 has been oxidised, but four protons have been pumped.
Cytochrome b and c
Cytochromes are small proteins containing a haem group (much like myoglobin or haemoglobin). They are grouped into three types (a, b and c) according to the type of haem and how it is bound into the protein.
Cytochrome-b proteins contain an iron protoporphyrin-IX prosthetic group, which is bound by dipole interactions. The Fe ion is hexacoordinated: 4 ligands from the N's of haem, and 2 from the histidines in the protein.
Cytochrome-c contains a haem-c prosthetic group bound covalently by its ring to cysteines in the protein. The Fe ion is hexacoordinated: 4 from the N's of haem, 1 from a histidine in the protein, and 1 from methionine in the protein.
Cytochrome oxidase dumps electrons from cytochrome-c onto oxygen, generating water, and pumping one proton per cytochrome-c.
Complex IV is more commonly termed cytochrome oxidase (or even just cyt-ox). It pumps 2 H+ per 2 cyt-cRED, and produces 1 H2O per 2 cyt-cRED oxidised. Complex IV receives its electrons from cytochrome-c, which is a small, mobile protein that diffuses from complex III to complex IV. The electrons are passed through a number of cytochrome-a and copper ion centres. CuB and cyt-a3 actually perform the reduction of oxygen to water. Each NADH originally oxidised yields 2 electrons, and these are enough to reduce half an O2 molecule to H2O (i.e. four electrons - two NADH - are required to reduce a whole molecule of dioxygen).
Cytochrome-a contains a haem-a prosthetic group bound by 'hydrophobic forces' to the protein. It also has a long phytol tail (just like chlorophyll). The Fe ion is pentacoordinated: 4 from the N's of haem, 1 from a histidine in the protein. This leaves a binding site for oxygen.
The reason that electrons flow through the various complexes is that earlier stages have lower redox potentials, so can provide electrons for downstream reactions.
The rotation of F0 can be seen by attaching a fluorescent actin tail to the F0 subunits.
Complex V is ATP synthase (an F-type ATPase). It converts an H+ gradient into ATP, producing c. 1 ATP per 3 or 4 H+ (stoichiometry still not quite certain). It actually acts like a motor: the FO subunit rotates as protons flow through and ATP is synthesised due to the conformational changes this causes in F1. It probably requires 3 protons to actually form one molecule of ATP, but one further proton is required to translocate ATP out of (and ADP/phosphate into) the matrix.
As protons flow through the a/b subunits (the stator) of FO, they force the ring of twelve c subunits (the rotor) in the membrane to rotate. This rotation is transmitted to the γ/ε subunits (the stalk) of F1, which change the conformation of the α/β subunits (the headpiece) of F1, makinf ADP and phosphate react to form ATP inside the β subunits. The headpiece is prevented from rotating by the binding of δ to the a/b stator, which is itself firmly anchored in the membrane.
The electron transport chain carries the electrons produced by the oxidation of NADH to NAD through complexes I, III and IV. This electron transport is used to drive proton pumping through the membrane. The electrons are eventually dumped onto oxygen, which is reduced to water. The proton gradient built up by these processes is used to drive the FOF1 ATPase (in reverse) to generate ATP. The oxidation of 1 NADH pumps (about) 10 protons. ATPase generates (about) 1 ATP from 4 protons.
2 ATP (substrate level phosphorylation)
2 ATP (substrate level phosphorylation)
2 NADH → 0 ATP
2 NADH → 5 ATP
2 ATP/GTP (substrate level phosphorylation)
8 NADH → 20 ATP
2 FADH2 (succinate) → 3 ATP
Approximate total yield
Take the energy budgets with a pinch of salt.
· NADH must be translocated into the mitochondrion from the cytoplasm: this costs ATP.
· The complexes are not 100% efficient.
· The inner membrane is not 100% impermeable.
· There is still argument about the stoichiometry of the various complexes.
Aerobic respiration is approximately 15 times more efficient than anaerobic. The P/O ratio (ATP made per oxygen atom reduced) is about 3 for NADH and 2 for succinate (FADH2). In books, you will find many different estimates of the ATP to glucose ratio, the number of protons pumped by each complex, the proton to ATP ratio for ATPase, etc. The numbers presented here are not to be taken as the definitive version!
Chemiosmosis is the name given to the generation of ATP from a proton gradient. It occurs in all living things:
The components of the chemiosmotic systems are similar too:
· Mitochondria, chloroplasts and purple-bacteria use a cytochrome complex to run the Q-cycle on quinones.
· The prosthetic group of cytochromes and chlorophyll are both porphyrins with phytanol tails.
Many of the proteins also contain
· Chloroplasts transfer electrons from water to NADPH, mitochondria transfer electrons from NADH to oxygen.
· All use an F-type ATPase in reverse to generate ATP.
Chemiosmosis can be disrupted by a variety of chemicals. In oxidative phosphorylation, some of these inhibitors are quite infamous:
Rotenone blocks electron flow from
· Antimycin-a blocks electron flow from cyt-bL to UQ in complex III (Q-cycle).
· Cyanide and carbon monoxide block access of O2 to cyt-a3 in complex IV.
· Oligomycin damages FO subunit of complex V.
These inhibitors were useful in the early research on the respiratory chain, and are still used to halt the chain at a particular point to study the stoichiometry of proton pumping.
Chemiosmosis works by generating a proton-motive force. The proton-motive force is the free energy associated with a gradient of protons across a proton-impermeable membrane. It is composed of two components: a chemical concentration gradient and an electrochemical charge gradient.
∆G = R T ln ( [H+]matrix ⁄ [H+]ims) − z F ∆Em
As well as simple inhibition, we can also uncouple electron transport from ATP synthesis by destroying the proton motive force. This is called uncoupling. Ionophores are rather good at this.
Dinitrophenol (DNP) is a proton ionophore (a weak acid). It carries protons across a membrane in a similar way to valinomycin with potassium ions. Pentachlorophenol (PCP) acts in a similar way to DNP. It was widely used as a biocide, especially in pallet board manufacture as a fungicide, but is now banned by the Biocidal Products Directive, because of its extreme toxicity and environmental persistence.
Other ionophores are more specific. Valinomycin is a potassium ionophore: it destroys ∆Em but not ∆pH: It uncouples ATP synthesis in mitochondria but not in chloroplasts, indicating that mitochondria use ∆Em (−150 mV), but not ∆pH (usually only about 0.5 pH units). Nigericin is an antiport ionophore that swaps H+ for K+. This is charge-neutral, so destroys ∆pH but has no effect on ∆Em. Nigericin effectively uncouples chloroplast ATP synthesis, but not mitochondrial, indicating that chloroplasts use ∆pH (usually about 4 units: stroma at pH 8, lumen at pH 4), but not ∆Em (0 mV).
Sometimes, organisms want to generate heat rather than ATP from chemiosmotic gradients. Brown fat tissue is mitochondria-rich adipose tissue. Lipid is oxidised and a proton gradient built up, but this is uncoupled from ATP synthesis by thermogenin. Thermogenin is a proton channel found in brown fat mitochondria. The flow of protons through the membrane generates heat.
Plants have a number of interesting 'extras' in their mitochondrial membranes. They have an intermembrane-space side NADPH dehydrogenase and a matrix-side rotenone-insensitive NADH dehydrogenase. They also have an alternative oxidase (alt-ox) that uncouples electron transport from ATP synthesis. This system (in theory) could completely uncouple NADH oxidation from ATP production, generating almost nothing but heat from the Krebs cycle.
What is the alternative oxidase for? It is known to be under hormonal control; it is stimulated by the plant hormone salicylic acid (Aspirin). It produces heat and could allows Krebs cycle (and the associated amino-acid pathways) to run even if ATP is not required by the cell (the energy overspill hypothesis). It also removes oxygen and prevents the build-up of reactive oxygen species produced by respiration and photosynthesis. However, in most cases, no-one really knows what it is 'for'. However, in aroids (arum lilies) it is know that the alternative oxidase generates heat in their inflorescence, volatilising amines and other fly-attracting chemicals. Skunk cabbage uses the heat generated to escape snow burial of its flowers.