Anaerobic and aerobic oxidation of glucose.
Alternative ways of monosaccharides metabolism.
Foods high in carbohydrate include fruits, sweets, soft drinks, breads, pastas, beans, potatoes, bran, rice, and cereals. Carbohydrates are a common source of energy in living organisms; however, no carbohydrate is an essential nutrient in humans.
Carbohydrates are not necessary building blocks of other molecules, and the body can obtain all its energy from protein and fats.[ The brain and neurons generally cannot burn fat for energy, but use glucose or ketones. Humans can synthesize some glucose (in a set of processes known as gluconeogenesis) from specific amino acids, from the glycerol backbone in triglycerides and in some cases from fatty acids. Carbohydrate and protein contain 4 calories per gram, while fats contain 9 calories per gram. In the case of protein, this is somewhat misleading as only some amino acids are usable for fuel.
Organisms typically cannot metabolize all types of carbohydrate to yield energy. Glucose is a nearly universal and accessible source of calories. Many organisms also have the ability to metabolize other monosaccharides and Disaccharides, though glucose is preferred. In Escherichia coli, for example, the lac operon will express enzymes for the digestion of lactose when it is present, but if both lactose and glucose are present the lac operon is repressed, resulting in the glucose being used first. Polysaccharides are also common sources of energy. Many organisms can easily break down starches into glucose, however, most organisms cannot metabolize cellulose or other polysaccharides like chitin and arabinoxylans. These carbohydrates types can be metabolized by some bacteria and protists. Ruminants and termites, for example, use microorganisms to processcellulose
Even though these complex carbohydrates are not very digestible, they represent an important dietary element for humans, called dietary fiber. Fiber enhances digestion, among other benefits.
Based on the effects on risk of heart disease and obesity, the Institute of Medicine recommends that American and Canadian adults get between 45–65% of dietary energy from carbohydrates. The Food and Agriculture Organization and World Health Organization jointly recommend that national dietary guidelines set a goal of 55–75% of total energy from carbohydrates, but only 10% directly from sugars (their term for simple carbohydrates).
The biological significance of carbohydrates in living organisms
A carbohydrate is an organic compound that is composed of atoms of carbon, hydrogen and oxygen in a ratio of 1 carbon atom, 2 hydrogen atoms, and 1 oxygen atom. Some carbohydrates are relatively small molecules, the most important to us is glucose which has 6 carbon atoms. These simple sugars are called monosaccharides.
The primary function of carbohydrates is for short-term energy storage (sugars are for Energy). A secondary function is intermediate-term energy storage (as in starch for plants and glycogen for animals). Other carbohydrates are involved as structural components in cells, such as cellulose which is found in the cell walls of plants.
Two common Monosaccharides, (single sugars) Glucose and Fructose
Hooking two monosaccharides together forms a more complex sugar, such as the union of glucose and fructose to give sucrose, or common table sugar. Compounds such as sucrose are called Disaccharides (two sugars). Both monosaccharides and disaccharides are soluble in water.
Larger, more complex carbohydrates are formed by linking shorter units together to form long or very long sugar chains called Polysaccharides. Because of their size, these are often times not soluble in water. Many biologically important compounds such as starches and cellulose are Polysaccharides. Starches are used by plants, and glycogen by animals, to store energy in their numerous carbon-hydrogen bonds, while cellulose is an important compound that adds strength and stiffness to a plant's cell wall.
Sugars are most often found in the form of a "RING". The glucose molecule in the image above and the one in the image below (Glc) are really the same molecule, just arranged differently. The corners of the "stop sign" represent Carbon atoms even thought they are not labeled with a "C" (its chemistry shorthand). To form these rings, the Carbonyl (C=0) Carbon of the straight-chain form (above) forms a bond with the next to last Carbon in the chain, making the ring.
The image on the left shows two monosaccharides, Glucose and Galactose (Gal). Examine their structure and you will notice there is very little difference. Their molecular formulas, C6H1206, are even the same. Molecules with the same chemical formula, but different molecular structures are called Isomers.
The sugar subunits can be linked by the reaction, dehydration synthesis, to form larger molecules. The disaccharide, Sucrose, is formed from two monosaccharides, Glucose and Fructose.
The disaccharide Lactose is a dimer (two subunits) of Glucose and Galactose, the disaccharide Maltose is a dimer of Glucose.
Large polymers of sugars are called Carbohydrates.
Carbohydrates can be 100's of sugars long and either straight or branched. The
term Complex Carbohydrate, or sometimes even just Carbohydrate refers to
long chains of sugars. Three common types of complex carbo's we will examine
are: Starch, Cellulose, and Glycogen. All three are composed only of Glucose.
They differ only in the bonding arrangements between the Glucose subunits. Not
all complex carbs are composed of glucose alone, many have highly unusual
sugars in their chains.
Starch is a long (100's) polymer of Glucose molecules, where all the sugars are oriented in the same direction. Starch is one of the primary sources of calories for humans.
Cellulose is a long (100's) polymer of Glucose molecules. However the orientation of the sugars is a little different. In Cellulose, every other sugar molecule is "upside-down". This small difference in structure makes a big difference in the way we use this molecule.
Glycogen is another Glucose polymer. Glycogen is a stored energy source, found in the Liver and muscles of Humans. Glycogen is different from both Starch and Cellulose in that the Glucose chain is branched or "forked".
As we noted, one function of carbohydrates (such as sugars) is for Energy. A secondary function is intermediate-term energy storage (as in starch for plants and glycogen for animals). Often the energy content of sugars is used to justify downing a Snickers Bar (or two). On the other hand, complex carbs that must be broken down before the sugars can be used are thought of as "Slow" Energy. The simple sugars are gradually released over time, providing a slow but steady source of Energy.
Pastas and whole-grain breads contain complex carbohydrates, which are long strands of glucose molecules.
Nutritionists recommend that 55–60 percent of calories come from carbohydrates, and especially complex carbohydrates.
Functions of Carbohydrate
All animals derive the major portion of their food calories from the different types of Carbohydrates in their diets. Most of the energy for the metabolic activities of the cell in all organisms is derived from the oxidation of Carbohydrate. Important functions of Carbohydrate are that of storing food, acting as a framework in body, performs are listed below.
Carbohydrate functions as Bio Fuel
Carbohydrate functions as an energy source of the body and acts as Bio fuel.Step wise details for the process of production of energy are discussed below.
· Polysaccharides such as starch and glycogen are first hydrolyzed by enzymes to Glucose.
· Glucose is the transported from one cell to another by blood in case of animals and cell sap in case of plants.
· Glucose is then oxidized to produce carbon dioxide and water.
· Energy is released in this process which is used for functioning of the cells.
Carbohydrate functions as Primary Source of Energy
The process of production of energy by carbohydrates is described in above steps. Now it is important to note, that fats and proteins can also be burned to provide energy but carbohydrate functions as primary source of energy. Fats are only burned if there is non availability of carbohydrates. When fat is burned in absence of carbohydrates, toxic compounds like called ketone bodies are produced. Accumulation of these ketone bodies over long period causes a condition called Ketosis. In this condition blood becomes unable to carry oxygen properly and this can be fatal. Thus, one of important function of carbohydrate is help burn fat properly.
Carbohydrate functions as storage food
Different forms of Carbohydrate are stored in living organism as storage food.
· Polysaccharide starch acts as storage food for plants.
· Glycogen stored in liver and muscles acts as storage food for animals.
· Inulin acts as storage food of dahlias, onion and garlic.
Thus carbohydrate performs the function of storing food.
Carbohydrate functions as framework in body
Different Carbohydrates especially Polysaccharides act as framework in living organism.
· Cellulose forms cell wall of plant cell along with hemicelluloses and Pectin
· Chitin forms cell wall of fungal cell and exoskeleton of arthropods
· Peptidoglycan forms cell wall of bacteria and cyanobacteria.
Thus carbohydrates function as contributing material to the cellular structure.
Carbohydrate functions as Anticoagulant
Heparin is a polysaccharide (carbohydrate) which acts as anticoagulant and prevents intravascular clotting.
Carbohydrate functions as Antigen
Many antigens are glycoprotein (which contains oligosaccharide) in nature and give immunological properties to the blood.
Carbohydrate functions as Hormone
Many Hormones like FSH (Follicular Stimulating Hormone which takes part in ovulation in females) and LH (Leutinizing Hormone) are glycoprotein and help in reproductive processes.
Carbohydrates provide raw material for industry
Carbohydrates are an important component of many industries like textile, paper, lacquers and breweries.
Agar is polysaccharide used in culture media, laxative and food.
Cellulose acts as roughage of food. It stimulates peristalsis movement and secretion of digestive enzymes.
Hyaluronic acid found in between joints acts as synovial fluid and provides frictionless movement.
Carbohydrates have the general molecular formula CH2O, and thus were once thought to represent "hydrated carbon". However, the arrangement of atoms in carbohydrates has little to do with water molecules.
Digestion of carbohydrates: localization, types, role of enzymes.
In simple terms, our digestion system - from the mouth to the small intestine - is designed to break down disaccharides and polysaccharides into monosaccharides. This metabolism of carbohydrates is achieved through the secretion of a number of digestive enzymes into the gastrointestinal tract (especially in the duodenum) where they attack carbohydrates and gradually convert them into simple sugars like glucose so they can be absorbed into the blood. Digestive enzymes are like biological scissors - they chop long starch molecules into simpler ones.
The process of digesting carbohydrates begins in the mouth. Our saliva contains an enzyme called amylase that starts breaking down the more complex carbs into simpler types.
In the Stomach
Enzyme activity continues in the stomach, but slows down significantly as digestive acids are released into the stomach by the glands.
In the Small
Another version of amylase is secreted by the pancreas into the duodenum (first section of small intestine). This cuts down carbohydrates into simple sugars - maltose, lactose and sucrose. As the carbohydrate passes further into the intestine, the enzymes maltase, lactase and sucrase chop maltose, lactose and sucrose into smaller bits, more easily absorbed, which are eventually converted to glucose and absorbed through the intestinal walls into the bloodstream.
Glucose Metabolism By The Liver
After carbohydrates are duly broken down into glucose, in the duodenum and jejunum of the small intestine, the glucose is absorbed into the bloodstream and taken to the liver, where it is stored or distributed to cells throughout the body for energy. In this way, the liver regulates blood glucose levels to provide sufficient energy for the body. For example, excess glucose (a cause of hyperglycemia) is converted in the liver to glycogen (glycogenolysis) in response to the hormone insulin, and stored. Likewise, if blood sugar levels fall, (eg. between meals), the glycogen is re-converted to glucose (glycogenolysis) in response to messages conveyed by the hormone glucagon, to prevent hypoglycemia. If glycogen levels are exhausted, glucagon can trigger the formation of glucose from some amino acids (protein) or glycerol (fats) - a process called gluconeogenesis.
The primary organ responsible for the regulation of blood glucose levels is the liver. Blood glucose levels must be maintained in the range of 80-120 mg/100 ml. In order to accomplish this the liver is capable of taking up large amounts of glucose. The enzyme glucokinase is responsible for doing this. Glucokinase has a high Km for glucose and is not inhibited by the product of the reaction glucose 6-phosphate so even when the serum glucose level is high, glucokinase remains active. The glucose 6-phosphate formed in the liver is trapped there because the phosphorylated derivative cannot cross the plasma membrane. In addition, the enzyme phosphorylase a acts as the glucose sensor for the body and is allosterically inactivated by high glucose concentrations. The fate of glucose 6-phosphate is controlled by the levels of insulin and glucogon. The figure below compares the control of blood glucose levels by the liver after eating and after an overnight fast.
What Determines Speed of Carb Digestion
Generally speaking, the speed of digestion is determined by the chemical nature of the carbohydrate itself, and thus how "resistant" it is to the activity of the enzymes. A simple sugar is much less resistant than a starch, and is digested or metabolized much faster. Things that slow down digestion include: the presence of acid (from gastric juices or the food itself), and the presence of soluble fiber.
Fiber- What is fiber? What your parents used to call roughage, food companies now promote as fiber. Fiber is an undigestable complex carbohydrate found in plants. Fiber is not a single food or substance. Fiber in itself has no calories because the body cannot absorb it. Therefore, high fiber foods, such as fruits and vegetables, are low in fat and low in calories. Fiber can be divided into two categories according to their physical characteristics and effects on the body: Water insoluble and water soluble. Each form functions differently and provides different health benefits. Insoluble fibers, such as cellulose, hemicellulose and lignin, do not dissolve in water. Soluble fibers, such as gums and pectins, do dissolve in water. Dietary Fiber is composed of undigestable complex carbohydrates. There are two basic types of fiber. Soluble Fiber - Pectins, acidic sugars often found in fruits. Insoluble fiber - Cellulose is one example, these are often found in the body of plants and in seed coats (where they are also known as bran).
There's a lot of confusion surrounding carbohydrates: Are they good or bad for us? Is a low-carb diet a good way to lose weight?
You need carbohydrates to function. They are the body's primary energy source and used for both physical activity and normal body functions such as brain function, heartbeat, breathing and digestion.
To eat or not to eat? There's a lot of confusion surrounding carbohydrates.
There is a huge difference between the natural "good" carbs that our bodies need to function and the unnatural, highly-processed, "refined" carbs so many of us eat every day!
The following food types are considered good carbohydrates: whole vegetables and fruits, beans, legumes and whole grains.
They include a range of the following healthy characteristics:
- High in fibre: These foods make you feel full, take longer to digest and cause a slow rise in blood sugar. They also promote waste elimination and help your body get rid of toxins. Good sources include raw fruits and vegetables with their skins, legumes, whole grains, berries and dried fruits.
- Low to moderate glycemic index: Good carbs stabilize blood sugar levels and insulin production.
- High in nutrients: They include natural vitamins, minerals and phytonutrients that promote good health
- Low calorie: They provide sustained energy, promote healthy weight loss and long-term weight maintenance
- Greater thermic-effect: They naturally stimulate metabolism and promote fat loss
Carbohydrates are bad for your skin
Bad carbs are refined, processed carbohydrate foods that have had all or most of their natural nutrients and fibre removed.
In addition to providing your body with empty calories, eating too many of these refined carbohydrates causes your blood sugar levels to spike, sending a signal to the pancreas to over-secrete insulin. In a nutshell, this facilitates the excess storage of fat.
And that's not all. A blood-sugar spike caused by refined carbs, followed by an over-secretion of insulin to combat the spike will result in an energy crash. This ultimately becomes a vicious cycle as you grab something sugary to try and bring your blood sugar levels back up. Bleached wheat flour, white sugar, artificial flavouring and preservatives are the most common ingredients used to make "bad carb" foods. Examples are the white versions of baked goods, breads and pastas, as well as snack foods, sugary cereals and soft drinks.
ESTIMATING HOW MANY CARBOHYDRATE CALORIES YOU SHOULD EAT
Carbohydrates contain four calories per gram. If you eat a 2,000-calorie-per-day diet and are aiming to eat 55 per cent of your calories from carbs, plan for 1,100 calories to come from carbs (275 grams).
Opt for whole grain versions of bread products, rice, pasta and cereals. Skip white sugar all together.
The mechanism of monosaccharides absorption
Simple sugars are far and away the predominant carbohydrate absorbed in the digestive tract, and in many animals the most important source of energy. Monosaccharides, however, are only rarely found in normal diets. Rather, they are derived by enzymatic digestion of more complex carbohydrates within the digestive tube.
Particularly important dietary carbohydrates include starch and disaccharides such as lactose and sucrose. None of these molecules can be absorbed for the simple reason that they cannot cross cell membranes unaided and, unlike the situation for monosaccharides, there are no transporters to carry them across.
This section will focus on understanding the processes involved in assimilation of three important carbohydrates: starch, lactose and sucrose. The key concepts involved in all three cases are that:
- the final enzymatic digestion that liberates monosaccharides is conducted by enzymes that are tethered in the lumenal plasma membrane of absorptive enterocytes (so-called "brush border hydrolyases").
- glucose generated by digestion of starch or lactose is absorbed in the small intestine only by cotransport with sodium, a fact that has exceptionally important implications in medicine.
Brush Border Hydrolases Generate Monosaccharides
Polysaccharides and disaccharides must be digested to monosaccharides prior to absorption and the key players in these processes are the brush border hydrolases, which include maltase, lactase and sucrase. Dietary lactose and sucrose are "ready" for digestion by their respective brush border enzymes. Starch, as discussed previously, is first digested to maltose by amylase in pancreatic secretions and, in some species, saliva.
Dietary lactose and sucrose, and maltose derived from digestion of starch, diffuse in the small intestinal lumen and come in contact with the surface of absorptive epithelial cells covering the villi where they engage with brush border hydrolases:
- maltase cleaves maltose into two molecules of glucose
- lactase cleaves lactose into a glucose and a galactose
- sucrase cleaves sucrose into a glucose and a fructose
At long last, we're ready to actually absorb these monosaccharides. Glucose and galactose are taken into the enterocyte by cotransport with sodium using the same transporter. Fructose enters the cell from the intestinal lumen via facilitated diffusion through another transporter.
Absorption of Glucose and Other Monosaccharides: Transport Across the Intestinal Epithelium
Absorption of glucose entails transport from the intestinal lumen, across the epithelium and into blood. The transporter that carries glucose and galactose into the enterocyte is the sodium-dependent hexose transporter, known more formally as SGLUT-1. As the name indicates, this molecule transports both glucose and sodium ion into the cell and in fact, will not transport either alone.
The essence of transport by the sodium-dependent hexose transporter involves a series of conformational changes induced by binding and release of sodium and glucose, and can be summarized as follows:
1. the transporter is initially oriented facing into the lumen - at this point it is capable of binding sodium, but not glucose
2. sodium binds, inducing a conformational change that opens the glucose-binding pocket
3. glucose binds and the transporter reorients in the membrane such that the pockets holding sodium and glucose are moved inside the cell
4. sodium dissociates into the cytoplasm, causing glucose binding to destabilize
5. glucose dissociates into the cytoplasm and the unloaded transporter reorients back to its original, outward-facing position
The animation seen below depicts digestion of maltose and entry of the resulting glucose, along with sodium, into the enterocyte (actually, two sodium ions are transported for each glucose). Despite the simplicity of the diagram, you should easily be able to identify the sodium-dependent hexose transporter and "watch" its conformational changes. Also, imagine the corresponding process involving lactose and sucrose assimilation.
Fructose is not co-transported with sodium. Rather it enters the enterocyte by another hexose transporter (GLUT5).
Once inside the enterocyte, glucose and sodium must be exported from the cell into blood. We've seen previously how sodium is rapidly shuttled out in exchange for potassium by the battery of sodium pumps on the basolateral membrane, and how that process maintains the electrochemical gradient across the epithelium. The energy stored in this gradient is actually what is driving glucose entry through the sodium-dependent hexose transporter described above. Recall also how the massive transport of sodium out of the cell establishes the osmotic gradient responsible for absorption of water.
Glucose, galactose and fructose are tranported out of the enterocyte through another hexose transporter (called GLUT-2) in the basolateral membrane. These monosaccharides then diffuse "down" a concentration gradient into capillary blood within the villus.
Decomposition of glucose in anaerobic conditions (glycolysis):
The Energy Derived from Glucose Oxidation
Aerobic glycolysis of glucose to pyruvate, requires two equivalents of ATP to activate the process, with the subsequent production of four equivalents of ATP and two equivalents of NADH. Thus, conversion of one mole of glucose to two moles of pyruvate is accompanied by the net production of two moles each of ATP and NADH.
Glucose + 2 ADP + 2 NAD+ + 2 Pi -----> 2 Pyruvate + 2 ATP + 2 NADH + 2 H+
generated during glycolysis is used to fuel mitochondrial ATP synthesis via oxidative
phosphorylation, producing either two or three equivalents of ATP
depending upon whether the glycerol
phosphate shuttle or the malate-aspartate
shuttle is used to transport the electrons from cytoplasmic NADH into
the mitochondria. The net yield from the oxidation of 1 mole of glucose to 2
moles of pyruvate is, therefore, either 6 or 8 moles of ATP. Complete oxidation
of the 2 moles of pyruvate, through the TCA
cycle, yeilds an additional 30 moles of ATP; the total yield, therefore
being either 36 or 38 moles of ATP from the complete oxidation of 1 mole of
glucose to CO2 and H2O.
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The Individual Reactions of Glycolysis
The pathway of glycolysis can be seen as consisting of 2 separate phases. The first is the chemical priming phase requiring energy in the form of ATP, and the second is considered the energy-yielding phase. In the first phase, 2 equivalents of ATP are used to convert glucose to fructose 1,6-bisphosphate (F1,6BP). In the second phase F1,6BP is degraded to pyruvate, with the production of 4 equivalents of ATP and 2 equivalents of NADH.
Pathway of glycolysis from glucose to pyruvate. Substrates and products are in blue, enzymes are in green. The two high energy intermediates whose oxidations are coupled to ATP synthesis are shown in red (1,3-bisphosphoglycerate and phosphoenolpyruvate).
The ATP-dependent phosphorylation of glucose to form glucose 6-phosphate (G6P)is the first reaction of glycolysis, and is catalyzed by tissue-specific isoenzymes known as hexokinases. The phosphorylation accomplishes two goals: First, the hexokinase reaction converts nonionic glucose into an anion that is trapped in the cell, since cells lack transport systems for phosphorylated sugars. Second, the otherwise biologically inert glucose becomes activated into a labile form capable of being further metabolized.
Four mammalian isozymes of hexokinase are known (Types I - IV), with the Type IV isozyme often referred to as glucokinase. Glucokinase is the form of the enzyme found in hepatocytes. The high Km of glucokinase for glucose means that this enzyme is saturated only at very high concentrations of substrate.
Comparison of the activities of hexokinase and glucokinase. The Km for hexokinase is significantly lower (0.1mM) than that of glucokinase (10mM). This difference ensures that non-hepatic tissues (which contain hexokinase) rapidly and efficiently trap blood glucose within their cells by converting it to glucose-6-phosphate. One major function of the liver is to deliver glucose to the blood and this in ensured by having a glucose phosphorylating enzyme (glucokinase) whose Km for glucose is sufficiently higher that the normal circulating concentration of glucose (5mM).
This feature of hepatic glucokinase allows the liver to buffer blood glucose. After meals, when postprandial blood glucose levels are high, liver glucokinase is significantly active, which causes the liver preferentially to trap and to store circulating glucose. When blood glucose falls to very low levels, tissues such as liver and kidney, which contain glucokinases but are not highly dependent on glucose, do not continue to use the meager glucose supplies that remain available. At the same time, tissues such as the brain, which are critically dependent on glucose, continue to scavenge blood glucose using their low Km hexokinases, and as a consequence their viability is protected. Under various conditions of glucose deficiency, such as long periods between meals, the liver is stimulated to supply the blood with glucose through the pathway of gluconeogenesis. The levels of glucose produced during gluconeogenesis are insufficient to activate glucokinase, allowing the glucose to pass out of hepatocytes and into the blood.
The regulation of hexokinase and glucokinase activities is also different. Hexokinases I, II, and III are allosterically inhibited by product (G6P) accumulation, whereas glucokinases are not. The latter further insures liver accumulation of glucose stores during times of glucose excess, while favoring peripheral glucose utilization when glucose is required to supply energy to peripheral tissues.
The second reaction of glycolysis is an isomerization, in which G6P is converted to fructose 6-phosphate (F6P). The enzyme catalyzing this reaction is phosphohexose isomerase (also known as phosphoglucose isomerase). The reaction is freely reversible at normal cellular concentrations of the two hexose phosphates and thus catalyzes this interconversion during glycolytic carbon flow and during gluconeogenesis.
6-Phosphofructo-1-Kinase (Phosphofructokinase-1, PFK-1):
The next reaction of glycolysis involves the utilization of a second ATP to convert F6P to fructose 1,6-bisphosphate (F1,6BP). This reaction is catalyzed by 6-phosphofructo-1-kinase, better known as phosphofructokinase-1 or PFK-1. This reaction is not readily reversible because of its large positive free energy (G0' = +5.4 kcal/mol) in the reverse direction. Nevertheless, fructose units readily flow in the reverse (gluconeogenic) direction because of the ubiquitous presence of the hydrolytic enzyme, fructose-1,6-bisphosphatase (F-1,6-BPase).
The presence of these two enzymes in the same cell compartment provides an example of a metabolic futile cycle, which if unregulated would rapidly deplete cell energy stores. However, the activity of these two enzymes is so highly regulated that PFK-1 is considered to be the rate-limiting enzyme of glycolysis and F-1,6-BPase is considered to be the rate-limiting enzyme in gluconeogenesis.
Aldolase catalyses the hydrolysis of F1,6BP into two 3-carbon products: dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P). The aldolase reaction proceeds readily in the reverse direction, being utilized for both glycolysis and gluconeogenesis.
The two products of the aldolase reaction equilibrate readily in a reaction catalyzed by triose phosphate isomerase. Succeeding reactions of glycolysis utilize G3P as a substrate; thus, the aldolase reaction is pulled in the glycolytic direction by mass action principals.
The second phase of glucose catabolism features the energy-yielding glycolytic reactions that produce ATP and NADH. In the first of these reactions, glyceraldehyde-3-P dehydrogenase (G3PDH) catalyzes the NAD+-dependent oxidation of G3P to 1,3-bisphosphoglycerate (1,3BPG) and NADH. The G3PDH reaction is reversible, and the same enzyme catalyzes the reverse reaction during gluconeogenesis.
The high-energy phosphate of 1,3-BPG is used to form ATP and 3-phosphoglycerate (3PG) by the enzyme phosphoglycerate kinase. Note that this is the only reaction of glycolysis or gluconeogenesis that involves ATP and yet is reversible under normal cell conditions. Associated with the phosphoglycerate kinase pathway is an important reaction of erythrocytes, the formation of 2,3-bisphosphoglycerate, 2,3BPG (see Figure below) by the enzyme bisphosphoglycerate mutase. 2,3BPG is an important regulator of hemoglobin's affinity for oxygen. Note that 2,3-bisphosphoglycerate phosphatase degrades 2,3BPG to 3-phosphoglycerate, a normal intermediate of glycolysis. The 2,3BPG shunt thus operates with the expenditure of 1 equivalent of ATP per triose passed through the shunt. The process is not reversible under physiological conditions.
The pathway for 2,3-bisphosphoglycerate (2,3-BPG) synthesis within erythrocytes. Synthesis of 2,3-BPG represents a major reaction pathway for the consumption of glucose in erythrocytes. The synthesis of 2,3-BPG in erythrocytes is critical for controlling hemoglobin affinity for oxygen. Note that when glucose is oxidized by this pathway the erythrocyte loses the ability to gain 2 moles of ATP from glycolytic oxidation of 1,3-BPG to 3-phosphoglycerate via the phosphoglycerate kinase reaction.
Phosphoglycerate Mutase and Enolase:
The remaining reactions of glycolysis are aimed at converting the relatively low energy phosphoacyl-ester of 3PG to a high-energy form and harvesting the phosphate as ATP. The 3PG is first converted to 2PG by phosphoglycerate mutase and the 2PG conversion to phosphoenoylpyruvate (PEP) is catalyzed by enolase
The final reaction of aerobic glycolysis is catalyzed by the highly regulated enzyme pyruvate kinase (PK). In this strongly exergonic reaction, the high-energy phosphate of PEP is conserved as ATP. The loss of phosphate by PEP leads to the production of pyruvate in an unstable enol form, which spontaneously tautomerizes to the more stable, keto form of pyruvate. This reaction contributes a large proportion of the free energy of hydrolysis of PEP.
Under aerobic conditions, pyruvate in most cells is further metabolized via the TCA cycle. Under anaerobic conditions and in erythrocytes under aerobic conditions, pyruvate is converted to lactate by the enzyme lactate dehydrogenase (LDH), and the lactate is transported out of the cell into the circulation. The conversion of pyruvate to lactate, under anaerobic conditions, provides the cell with a mechanism for the oxidation of NADH (produced during the G3PDH reaction) to NAD+; which occurs during the LDH catalyzed reaction. This reduction is required since NAD+ is a necessary substrate for G3PDH, without which glycolysis will cease. Normally, during aerobic glycolysis the electrons of cytoplasmic NADH are transferred to mitochondrial carriers of the oxidative phosphorylation pathway generating a continuous pool of cytoplasmic NAD+.
Aerobic glycolysis generates substantially more ATP per mole of glucose oxidized than does anaerobic glycolysis. The utility of anaerobic glycolysis, to a muscle cell when it needs large amounts of energy, stems from the fact that the rate of ATP production from glycolysis is approximately 100X faster than from oxidative phosphorylation. During exertion muscle cells do not need to energize anabolic reaction pathways. The requirement is to generate the maximum amount of ATP, for muscle contraction, in the shortest time frame. This is why muscle cells derive almost all of the ATP consumed during exertion from anaerobic glycolysis.
The reactions catalyzed by hexokinase, PFK-1 and PK all proceed with a relatively large free energy decrease. These nonequilibrium reactions of glycolysis would be ideal candidates for regulation of the flux through glycolysis. Indeed, in vitro studies have shown all three enzymes to be allosterically controlled.
Regulation of hexokinase, however, is not the major control point in glycolysis. This is due to the fact that large amounts of G6P are derived from the breakdown of glycogen (the predominant mechanism of carbohydrate entry into glycolysis in skeletal muscle) and, therefore, the hexokinase reaction is not necessary. Regulation of PK is important for reversing glycolysis when ATP is high in order to activate gluconeogenesis. As such this enzyme catalyzed reaction is not a major control point in glycolysis. The rate limiting step in glycolysis is the reaction catalyzed by PFK-1.
PFK-1 is a tetrameric enzyme that exist in two conformational states termed R and T that are in equilibrium. ATP is both a substrate and an allosteric inhibitor of PFK-1. Each subunit has two ATP binding sites, a substrate site and an inhibitor site. The substrate site binds ATP equally well when the tetramer is in either conformation. The inhibitor site binds ATP essentially only when the enzyme is in the T state. F6P is the other substrate for PFK-1 and it also binds preferentially to the R state enzyme. At high concentrations of ATP, the inhibitor site becomes occupied and shifting the equilibrium of PFK-1 comformation to that of the T state decreasing PFK-1's ability to bind F6P. The inhibition of PFK-1 by ATP is overcome by AMP which binds to the R state of the enzyme and, therefore, stabilizes the conformation of the enzyme capable of binding F6P. The most important allosteric regulator of both glycolysis and gluconeogenesis is fructose 2,6-bisphosphate, F2,6BP, which is not an intermediate in glycolysis or in gluconeogenesis.
Regulation of glycolysis and gluconeogenesis by fructose 2,6-bisphosphate (F2,6BP). The major sites for regulation of glycolysis and gluconeogenesis are the phosphofructokinase-1 (PFK-1) and fructose-1,6-bisphosphatase (F-1,6-BPase) catalyzed reactions. PFK-2 is the kinase activity and F-2,6-BPase is the phosphatase activity of the bi-functional regulatory enzyme, phosphofructokinase-2/fructose-2,6-bisphosphatase. PKA is cAMP-dependent protein kinase which phosphorylates PFK-2/F-2,6-BPase turning on the phosphatase activity. (+ve) and (-ve) refer to positive and negative activities, respectively.
The synthesis of F2,6BP is catalyzed by the bifunctional enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase (PFK-2/F-2,6-BPase). In the nonphosphorylated form the enzyme is known as PFK-2 and serves to catalyze the synthesis of F2,6BP by phosphorylating fructose 6-phosphate. The result is that the activity of PFK-1 is greatly stimulated and the activity of F-1,6-BPase is greatly inhibited.
Under conditions where PFK-2 is active, fructose flow through the PFK-1/F-1,6-BPase reactions takes place in the glycolytic direction, with a net production of F1,6BP. When the bifunctional enzyme is phosphorylated it no longer exhibits kinase activity, but a new active site hydrolyzes F2,6BP to F6P and inorganic phosphate. The metabolic result of the phosphorylation of the bifunctional enzyme is that allosteric stimulation of PFK-1 ceases, allosteric inhibition of F-1,6-BPase is eliminated, and net flow of fructose through these two enzymes is gluconeogenic, producing F6P and eventually glucose.
The interconversion of the bifunctional enzyme is catalyzed by cAMP-dependent protein kinase (PKA), which in turn is regulated by circulating peptide hormones. When blood glucose levels drop, pancreatic insulin production falls, glucagon secretion is stimulated, and circulating glucagon is highly increased. Hormones such as glucagon bind to plasma membrane receptors on liver cells, activating membrane-localized adenylate cyclase leading to an increase in the conversion of ATP to cAMP (see diagram below). cAMP binds to the regulatory subunits of PKA, leading to release and activation of the catalytic subunits. PKA phosphorylates numerous enzymes, including the bifunctional PFK-2/F-2,6-BPase. Under these conditions the liver stops consuming glucose and becomes metabolically gluconeogenic, producing glucose to reestablish normoglycemia.
Representative pathway for the activation of cAMP-dependent protein kinase (PKA). In this example glucagon binds to its' cell-surface receptor, thereby activating the receptor. Activation of the receptor is coupled to the activation of a receptor-coupled G-protein (GTP-binding and hydrolyzing protein) composed of 3 subunits. Upon activation the alpha subunit dissociates and binds to and activates adenylate cyclase. Adenylate cylcase then converts ATP to cyclic-AMP (cAMP). The cAMP thus produced then binds to the regulatory subunits of PKA leading to dissociation of the associated catalytic subunits. The catalytic subunits are inactive until dissociated from the regulatory subunits. Once released the catalytic subunits of PKA phosphorylate numerous substrate using ATP as the phosphate donor.
Regulation of glycolysis also occurs at the step catalyzed by pyruvate kinase, (PK). The liver enzyme has been most studied in vitro. This enzyme is inhibited by ATP and acetyl-CoA and is activated by F1,6BP. The inhibition of PK by ATP is similar to the effect of ATP on PFK-1. The binding of ATP to the inhibitor site reduces its affinity for PEP. The liver enzyme is also controlled at the level of synthesis. Increased carbohydrate ingestion induces the synthesis of PK resulting in elevated cellular levels of the enzyme.
A number of PK isozymes have been described. The liver isozyme (L-type), characteristic of a gluconeogenic tissue, is regulated via phosphorylation by PKA, whereas the M-type isozyme found in brain, muscle, and other glucose requiring tissue is unaffected by PKA. As a consequence of these differences, blood glucose levels and associated hormones can regulate the balance of liver gluconeogenesis and glycolysis while muscle metabolism remains unaffected.
In erythrocytes, the fetal PK isozyme has much greater activity than the adult isozyme; as a result, fetal erythrocytes have comparatively low concentrations of glycolytic intermediates. Because of the low steady-state concentration of fetal 1,3BPG, the 2,3BPG shunt (see diagram above) is greatly reduced in fetal cells and little 2,3BPG is formed. Since 2,3BPG is a negative effector of hemoglobin affinity for oxygen, fetal erythrocytes have a higher oxygen affinity than maternal erythrocytes. Therefore, transfer of oxygen from maternal hemoglobin to fetal hemoglobin is favored, assuring the fetal oxygen supply. In the newborn, an erythrocyte isozyme of the M-type with comparatively low PK activity displaces the fetal type, resulting in an accumulation of glycolytic intermediates. The increased 1,3BPG levels activate the 2,3BPG shunt, producing 2,3BPG needed to regulate oxygen binding to hemoglobin.
Genetic diseases of adult erythrocyte PK are known in which the kinase is virtually inactive. The erythrocytes of affected individuals have a greatly reduced capacity to make ATP and thus do not have sufficient ATP to perform activities such as ion pumping and maintaining osmotic balance. These erythrocytes have a short half-life, lyse readily, and are responsible for some cases of hereditary hemolytic anemia.
The liver PK isozyme is regulated by phosphorylation, allosteric effectors, and modulation of gene expression. The major allosteric effectors are F1,6BP, which stimulates PK activity by decreasing its Km(app) for PEP, and for the negative effector, ATP. Expression of the liver PK gene is strongly influenced by the quantity of carbohydrate in the diet, with high-carbohydrate diets inducing up to 10-fold increases in PK concentration as compared to low carbohydrate diets. Liver PK is phosphorylated and inhibited by PKA, and thus it is under hormonal control similar to that described earlier for PFK-2.
Muscle PK (M-type) is not regulated by the same mechanisms as the liver enzyme. Extracellular conditions that lead to the phosphorylation and inhibition of liver PK, such as low blood glucose and high levels of circulating glucagon, do not inhibit the muscle enzyme. The result of this differential regulation is that hormones such as glucagon and epinephrine favor liver gluconeogenesis by inhibiting liver glycolysis, while at the same time, muscle glycolysis can proceed in accord with needs directed by intracellular conditions.
Pyruvate is the branch point molecule of glycolysis. The ultimate fate of pyruvate depends on the oxidation state of the cell. In the reaction catalyzed by G3PDH a molecule of NAD+ is reduced to NADH. In order to maintain the re-dox state of the cell, this NADH must be re-oxidized to NAD+. During aerobic glycolysis this occurs in the mitochondrial electron transport chain generating ATP. Thus, during aerobic glycolysis ATP is generated from oxidation of glucose directly at the PGK and PK reactions as well as indirectly by re-oxidation of NADH in the oxidative phosphorylation pathway. Additional NADH molecules are generated during the complete aerobic oxidation of pyruvate in the TCA cycle. Pyruvate enters the TCA cycle in the form of acetyl-CoA which is the product of the pyruvate dehydrogenase reaction. The fate of pyruvate during anaerobic glycolysis is reduction to lactate.
During anaerobic glycolysis, that period of time when glycolysis is proceeding at a high rate (or in anaerobic organisms), the oxidation of NADH occurs through the reduction of an organic substrate. Erythrocytes and skeletal muscle (under conditions of exertion) derive all of their ATP needs through anaerobic glycolysis. The large quantity of NADH produced is oxidized by reducing pyruvate to lactate. This reaction is carried out by lactate dehydrogenase, (LDH). The lactate produced during anaerobic glycolysis diffuses from the tissues and is transproted to highly aerobic tissues such as cardiac muscle and liver. The lactate is then oxidized to pyruvate in these cells by LDH and the pyruvate is further oxidized in the TCA cycle. If the energy level in these cells is high the carbons of pyruvate will be diverted back to glucose via the gluconeogenesis pathway.
cells contain two distinct types of LDH subunits, termed M and H. Combinations
of these different subunits generates LDH isozymes with different characteristics.
The H type subunit predominates in aerobic tissues such as heart muscle (as the
H4 tetramer) while the M subunit predominates in anaerobic tissues such as
skeletal muscle as the M4 tetramer). H4 LDH has a low Km for
pyruvate and also is inhibited by high levels of pyruvate. The M4 LDH enzyme
has a high Km for pyruvate and is not inhibited by pyruvate. This
suggsts that the H-type LDH is utilized for oxidizing lactate to pyruvate and
the M-type the reverse.
The content of glucose in blood:
Blood sugar concentration, or glucose level, refers to the amount of glucose present in a mammal's blood. Normally, the blood glucose level is maintained at a reference range between about 4 and 6 mM (mmol/l). It is tightly regulated in the human body. The normal blood glucose level is about 90mg/100ml, which works out to 5mM (mmol/l), since the molecular weight of glucose, C6H12O6, is about 180 g/mol daltons. The total amount of glucose in circulating blood is therefore about 3.3 to 7g (assuming an ordinary adult blood volume of 5 litres, plausible for an average adult male). Glucose levels rise after meals for an hour or two by a few grams and are usually lowest in the morning, before the first meal of the day. Transported via the bloodstream from the intestines or liver to body cells, Glucose is the primary source of energy for the body's cells.
Failure to maintain blood glucose in the normal range leads to conditions of persistently high (hyperglycemia) or low (hypoglycemia) blood sugar. Diabetes mellitus, characterized by persistent hyperglycemia from any of several causes, is the most prominent disease related to failure of blood sugar regulation
Causes and consequences of hypo- and hyperglycemia
Hyperglycemia, hyperglycaemia, or high blood sugar is a condition in which an excessive amount of glucose circulates in the blood plasma. This is generally a blood glucose level of 10+ mmol/L (180 mg/dl), but symptoms may not start to become noticeable until later numbers like 15-20+ mmol/L (270-360 mg/dl). However, chronic levels exceeding 125 mg/dl can produce organ damage.
The origin of the term is Greek: hyper-, meaning excessive; -glyc-, meaning sweet; and -emia, meaning "of the blood".
Hypoglycaemia or hypoglycemia is the medical term for a pathologic state produced by a lower than normal level of blood glucose. The term hypoglycemia literally means "under-sweet blood" (Gr. hypo-, glykys, haima). The term also refers to a putative condition that is scientifically disputed and which is perhaps more properly considered as a part of "alternative" medicine.[neutrality disputed] This is covered at the end of this article.
Hypoglycemia can produce a variety of symptoms and effects but the principal problems arise from an inadequate supply of glucose as fuel to the brain, resulting in impairment of function (neuroglycopenia). Derangements of function can range from vaguely "feeling bad" to coma, anymous seizures, and (rarely) permanent brain damage or death. Hypoglycemia can arise from many causes and can occur at any age. It also sometimes occurs at random.
The most common forms of moderate and severe hypoglycemia occur as a complication of treatment of diabetes mellitus treated with insulin or less frequently with certain oral medications. Hypoglycemia is usually treated by the ingestion or administration of dextrose, or foods quickly digestible to glucose.
Endocrinologists (specialists in hormones, including those which regulate glucose metabolism) typically consider the following criteria (referred to as Whipple's triad) as proving that individual's symptoms can be attributed to hypoglycemia:
1. Symptoms known to be caused by hypoglycemia
2. Low glucose at the time the symptoms occur
3. Reversal or improvement of symptoms or problems when the glucose is restored to normal
However, not everyone has accepted these suggested diagnostic criteria, and even the level of glucose low enough to define hypoglycemia has been a source of controversy in several contexts. For many purposes, plasma glucose levels below 70 mg/dl or 3.9 mmol/L are considered hypoglycemic; these issues are detailed below.
Biological and energetic value of glycogenolysis
In glycogenolysis, glycogen stored in the liver and muscles, is converted first to glucose-1- phosphate and then into glucose-6-phosphate. Two hormones which control glycogenolysis are a peptide, glucagon from the pancreas and epinephrine from the adrenal glands.
Glucagon is released from the pancreas in response to low blood glucose and epinephrine is released in response to a threat or stress. Both hormones act upon enzymes to stimulate glycogen phosphorylase to begin glycogenolysis and inhibit glycogen synthetase (to stop glycogenesis).
Glycogen is a highly branched polymeric structure containing glucose as the basic monomer. First individual glucose molecules are hydrolyzed from the chain, followed by the addition of a phosphate group at C-1. In the next step the phosphate is moved to the C-6 position to give glucose 6-phosphate, a cross road compound.
Glucose-6-phosphate is the first step of the glycolysis pathway if glycogen is the carbohydrate source and further energy is needed. If energy is not immediately needed, the glucose-6-phosphate is converted to glucose for distribution in the blood to various cells such as brain cells.
ІІ. Alternative ways of monosaccharides metabolism.
Entry of other carbohydrates (fructose, galactose) into the glycolytic sequence
Entry of galactose into the glycolytic-gluconeogenetic pathway.
Galactose comes from the splitting of lactose (galactose + glucose).
Lactose is broken
down by lactase, the sugars then freely enter the intestinal cells (not insulin
Galactose (like all monosaccharides) must be phosphorylated in order to enter the pathways.
In order for galactose to enter the glycolytic pathway is must first be converted to galactose-1-phosphate and then activated by adding UDP to make galactose-UDP.
Lack of the enzyme galactokinase.
- Leads to excess galactose in the blood
- Excess galactose in the blood causes galactosemia and galactosuria
- If a person with galactose intolerance has galactose in the diet it will be converted to galactitol
The accumulation of galactitol will damage the eyes (cataract)
Hepatic metabolism of fructose.
The diatery source of fructose is the disaccharide sucrose (glucose+fructose).
FK = Fructokinase
GK/HK = Glucokinase / Hexokinase ( either one catalyzes this reaction, although they both have a much higher affinity for Glucose, they are able to process Fructose)
A = Aldolase A
B = Aldolase B
The minor pathway
is that of Fructose => Fructose-6-Phosphate (F-6-P)
The major pathway is that of Fructose => Fructose-1-Phosphate (F-1-P)
The regulatory/toxic effects of fructose-1-phosphate.
rapidly, forming Fructose-1-phosphate, but Aldolase B doesn't work as fast.
This increases the amount of Fructose-1-phosphate leading to an accumulation, especially in the liver (since the liver is the major organ for fructose metabolism).
Phosphate is bound to fructose, so the level of ATP decreases. This is called phosphate trapping .
Alcoholic fermentation. Common reactions for fermentation and glycolysis.
Difference of these processes.
When the oxygen supply runs short in heavy or prolonged exercise, muscles obtain most of their energy from an anaerobic (without oxygen) process called glycolysis. Yeast cells obtain energy under anaerobic conditions using a very similar process called alcoholic fermentation. Glycolysis is the chemical breakdown of glucose to lactic acid. This process makes energy available for cell activity in the form of a high-energy phosphate compound known as adenosine triphosphate (ATP). Alcoholic fermentation is identical to glycolysis except for the final step (Fig. 1). In alcoholic fermentation, pyruvic acid is broken down into ethanol and carbon dioxide. Lactic acid from glycolysis produces a feeling of tiredness; the products of alcoholic fermentation have been used in baking and brewing for centuries.
Both alcoholic fermentation and glycolysis are anaerobic fermentation processes that begin with the sugar glucose. Glycolysis requires 11 enzymes which degrade glucose to lactic acid (Fig. 2). Alcoholic fermentation follows the same enzymatic pathway for the first 10 steps. The last enzyme of glycolysis, lactate dehydrogenase, is replaced by two enzymes in alcoholic fermentation. These two enzymes, pyruvate decarboxylase and alcoholic dehydrogenase, convert pyruvic acid into carbon dioxide and ethanol in alcoholic fermentation.
The most commonly accepted evolutionary scenario states that organisms first arose in an atmosphere lacking oxygen. Anaerobic fermentation is supposed to have evolved first and is considered the most ancient pathway for obtaining energy. There are several scientific difficulties, however, with considering fermentations as primitive energy harvesting mechanisms produced by time and chance.
First of all, it takes ATP energy to start the process that will only later generate a net gain in ATP. Two ATPs are put into the glycolytic pathway for priming the reactions, the expenditure of energy by conversion of ATP to ADP being required in the first and third steps of the pathway (Fig. 2). A total of four ATPs are obtained only later in the sequence, making a net gain of two ATPs for each molecule of glucose degraded. The net gain of two ATPs is not realized until the tenth enzyme in the series catalyzes phosphoenolpyruvate to ATP and pyruvic acid (pyruvate). This means that neither glycolysis nor alcoholic fermentation realizes any gain in energy (ATP) until the tenth enzymatic breakdown.
It is purely wishful thinking to suppose that a series of 10 simultaneous, beneficial, additive mutations could produce 10 complex enzymes to work on 10 highly specific substances and that these reactions would occur in sequence. Enzymes are proteins consisting of amino acids united in polypeptide chains. Their complexity may be illustrated by the enzyme glyceraldehyde phosphate dehydrogenase, which is the enzyme that catalyzes the oxidation of phosphoglyceraldehyde in glycolysis and alcoholic fermentation. Glyceraldehyde phosphate dehydrogenase consists of four identical chains, each having 330 amino acid residues. The number of different possible arrangements for the amino acid residues of this enzyme is astronomical.
To illustrate, let us consider a simple protein containing only 100 aim acids. There are 20 different kinds of L-amino acids in proteins, and each can be used repeatedly in chains of 100. Therefore, they could be arranged in 20100 or 10130 different ways. Even if a hundred million billion of these (1017) combinations could function for a given purpose, there is only one chance in 10113 of getting one of these required amino acid sequences in a small protein consisting of 100 amino acids.
Fig. 2. Notice that ATP is formed at two different locations above (steps 7 & 10). Because there are 2 molecules of the substrates, there will be 2 molecules of ATP formed at both locations, making a total of 4 molecules of ATP. Two molecules of ATP were necessary for priming the original breakdown of glucose (step 1). Therefore, a net of 2 molecules of ATP are recognized from the entire breakdown of glucose pyruvate. (4 ATP formed - 2 ATP primers = 2 ATP net overall gain.) Notice also that this MW net gain In ATP is not recognized until phosphoenolpyruvate is broken down by pyruvate kinase to form 2 molecules of pyruvate. This means that 10 enzymatic reactions must proceed in sequence, before energy in the form of ATP is obtained.
There are still other problems with the theory of evolution for alcoholic fermentation and glycolytic pathways. It is necessary to account for the numerous complex regulatory mechanisms which control these chemical pathways. For example, phosphofructokinase is a regulatory enzyme which limits the rate of glycolysis. Glycogen phosphorylase is also a regulatory enzyme; it converts glycogen to glucose-1-phosphate and thus makes glycogen available for glycolytic breakdown. In complex organisms there are several hormones such as somatotropin, insulin, glucagon, glucocorticoids, adrenaline thyroxin and a host of others which control utilization of glucose. No evolutionary mechanism has ever been proposed to account for these control mechanisms.
In addition to the regulators, complex cofactors are absolutely essential for glycolysis. One of the two key ATP energy harvesting steps in glycolysis requires a dehydrogenase enzyme acting in concert with the "hydrogen shuttle" redox reactant, nicotinamide adenine dinucleotide (NAD+). To keep the reaction sequence going, the reduced cofactor (NADH + H +) must be continuously regenerated by steps later in the sequence (Fig. 2), and that requires one enzyme in glycolysis (lactic dehydrogenase) and another (alcohol dehydrogenase) in alcoholic fermentation. In the absence of continuously cycled NAD+, "simple" anaerobic ATP energy harvest would be impossible.
And there are further difficulties yet for evolutionary theory to surmount. At one point, an intermediate in the glycolytic pathway is "stuck" with a phosphate group (needed to make ATP) in the low energy third carbon position. A remarkable enzyme, a "mutase" (Step 8), shifts the phosphate group to the second carbon position—but only in the presence of pre-existent primer amounts of an extraordinary molecule, 2,3-diphosphoglyceric acid. Actually, the shift of the phosphate from the third to the second position using the "mutase" and these "primer" molecules accomplishes nothing notable directly, but it "sets up" the ATP energy-harvesting reaction which occurs two steps later!
In summary, the following items make an evolutionary origin for glycolysis and alcoholic fermentation totally untenable: (1) the extreme improbability of getting even one simple enzyme by random processes; (2) the fact that the overall net gain in energy (ATP) is not recognized until pyruvate formation suggests that the chemical reaction must proceed through at least 10 enzymatic steps and that these steps of necessity must be in sequence; (3) the complex regulatory mechanisms, cofactors, and "primers" necessary for glucose utilization cannot be explained by evolutionary speculation.
On the other hand, the tight fit among complex and interdependent steps—especially the way some reactions take on meaning only in terms of reactions that occur much later in the sequence—seems to point clearly to creation with a teleological purpose, by an Intelligence and Power far greater than man's.
The principle of lactic acid amount measurement in blood serum, diagnostic significance.
During intense exercise, muscle and blood lactate can rise to very high levels. This accumulation above resting levels represents the balance of production and removal. It says nothing about whether accumulation is due to an increased rate of production or decreased rate of removal, or both. Similarly, if lactate concentrations in the blood do not rise above resting levels during or immediately following exercise, it also infers nothing about lactate or lactic acid production during that activity. It may be that lactic acid production is several times higher than at rest but that it is matched by its removal showing no net increase.
A common misinterpretation is that blood lactate or even lactic acid, has a direct detrimental effect on muscle performance. However, most researchers agree that any negative effect on performance associated with blood lactate accumulation is due to an increase in hydrogen ions. When lactic acid dissociates it forms lactate and hydrogen ions - which leads to an increase in acidity. So it is not accurate to blame either lactate or lactic acid for having a direct negative impact on muscular performance.
The increase in hydrogen ions and subsequent acidity of the internal environment is called acidosis.
So this unfavourable acidosis is the result of an increased concentration or accumulation of hydrogen ions. It may seem logical to conclude then, that any increase in production of lactic acid and hence lactate is detrimental as it will increase the production of hydrogen ions. However, accumulation is the key term here as an increased production of hydrogen ions (due to an increase production of lactic acid) will have no detrimental effect if clearance is just takes it a step further…
They suggest that lactate production (especially if accompanied by a high capacity for lactate removal) may be more likely to delay the onset of acidosis. The reasons for this, amongst others, are that lactate serves to consume hydrogen ions and allows the transport of hydrogen ions from the cell. Similarly, they maintain, there is a wealth of research evidence to show that acidosis is caused by reactions other than lactate production. Rogers et al. do conclude however, that increased lactate concentration, although not causative, coincides with cellular acidosis and remains a good indirect marker for the onset of fatigue.
Acidosis and Fatigue
As mentioned earlier, there has been substantial research to show that an increase concentration of hydrogen ions and a decrease in pH (increase in acidity) within muscle or plasma, causes fatigue. Additionally, induced acidosis can impair muscle contractility even in non-fatigued humans and several mechanisms to explain such effects have been provided.
Yet in the last 10 years a number of high profile papers have challenged even this most basic assumption of fatigue. A 2006 review of these by Cairns suggests that experiments on isolated muscle show that acidosis has little detrimental effect or may even improve muscle performance during high-intensity exercise.
In place of acidosis it may be inorganic phosphate that is major cause of muscle fatigue. Recall that an inorganic phosphate is produced during the breakdown of ATP to ADP. However, there are several limitations regarding this phosphate hypothesis. Another proposal for a major contributor to fatigue, rather than acidosis, is the accumulation of potassium ions in muscle interstitium.
Contrary to this new research (which is by no means definitive) is the argument that if acidosis plays no role in fatigue then it is surprising that alkalosis (through sodium bicarbonate consumption for example) can improve exercise performance in events lasting 1-10 minutes. To reconcile this, Cairns (18) hypothesizes that while acidosis has little detrimental effect or may even improve muscle performance in isolated muscle, severe blood plasma acidosis may impair performance by causing a reduced central nervous system drive to muscle.
Pentose phosphate pathway of carbohydrates metabolism
The pentose phosphate pathway is primarily an anabolic pathway that utilizes the 6 carbons of glucose to generate 5 carbon sugars and reducing equivalents. However, this pathway does oxidize glucose and under certain conditions can completely oxidize glucose to CO2 and water. The primary functions of this pathway are:
1. To generate reducing equivalents, in the form of NADPH, for reductive biosynthesis reactions within cells.
2. To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids.
3. Although not a significant function of the PPP, it can operate to metabolize dietary pentose sugars derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary carbohydrates into glycolytic/gluconeogenic intermediates.
Enzymes that function primarily in the reductive direction utilize the NADP+/NADPH cofactor pair as co-factors as opposed to oxidative enzymes that utilize the NAD+/NADH cofactor pair. The reactions of fatty acid biosynthesis and steroid biosynthesis utilize large amounts of NADPH. As a consequence, cells of the liver, adipose tissue, adrenal cortex, testis and lactating mammary glan have high levels of the PPP enzymes. In fact 30% of the oxidation of glucose in the liver occurs via the PPP. Additionally, erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH used in the reduction of glutathione (see below). The conversion of ribonucleotides to deoxyribonucleotides (through the action of ribonucleotide reductase) requires NADPH as the electron source, therefore, any rapidly proliferating cell needs large quantities of NADPH.
Reactions of the Pentose Phosphate Pathway
The reactions of the PPP operate exclusively in the cytoplasm. From this perspective it is understandable that fatty acid synthesis (as opposed to oxidation) takes place in the cytoplasm. The pentose phosphate pathway has both an oxidative and a non-oxidative arm. The oxidation steps, utilizing glucose-6-phosphate (G6P) as the substrate, occur at the beginning of the pathway and are the reactions that generate NADPH. The reactions catalyzed by glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase generate one mole of NADPH each for every mole of glucose-6-phosphate (G6P) that enters the PPP.
The non-oxidative reactions of the PPP are primarily designed to generate R5P. Equally important reactions of the PPP are to convert dietary 5 carbon sugars into both 6 (fructose-6-phosphate) and 3 (glyceraldehyde-3-phosphate) carbon sugars which can then be utilized by the pathways of glycolysis.
The primary enzymes involved in the non-oxidative steps of the PPP are transaldolase and transketolase:
Transketolase functions to transfer 2 carbon groups from substrates of the PPP, thus rearranging the carbon atoms that enter this pathway. Like other enzymes that transfer 2 carbon groups, transketolase requires thiamine pyrophosphate (TPP) as a co-factor in the transfer reaction.
Transaldolase transfers 3 carbon groups and thus is also involved in a rearrangement of the carbon skeletons of the substrates of the PPP. The transaldolase reaction involves Schiff base formation between the substrate and a lysine residue in the enzyme.
The net result of the PPP, if not used solely for R5P production, is the oxidation of G6P, a 6 carbon sugar, into a 5 carbon sugar. In turn, 3 moles of 5 carbon sugar are converted, via the enzymes of the PPP, back into two moles of 6 carbon sugars and one mole of 3 carbon sugar. The 6 carbon sugars can be recycled into the pathway in the form of G6P, generating more NADPH. The 3 carbon sugar generated is glyceraldehyde-3-phsphate which can be shunted to glycolysis and oxidized to pyruvate. Alternatively, it can be utilized by the gluconeogenic enzymes to generate more 6 carbon sugars (fructose-6-phosphate or glucose-6-phosphate).
Metabolic Disorders Associated with the PPP
Diabetes, Carbohydrate-Modified Diets, and Carbohydrate Counting
Diabetes is a condition that alters the way the body handles carbohydrates. In terms of diet modifications, diabetics can control blood sugar levels by appropriately managing the carbohydrates, proteins, and fats in their meals. The amount of carbohydrates, not necessarily the source, is the primary issue. Blood glucose levels after a meal can be related to the process of food preparation, the amount of food eaten, fat intake, sugar absorption, and the combination of foods in the meal or snack.
One method of monitoring carbohydrate levels—carbohydrate counting—assigns a certain number of carbohydrate grams or exchanges to specific foods. Calculations are used to determine insulin need, resulting in better control of blood glucose levels with a larger variety of foods. Overall, diabetic diets can include moderate amounts of sugar, as long as they are carefully monitored.
Oxidative stress within cells is controlled primarily by the action of the peptide, glutathione, GSH. See Specialized Products of Amino Acids for the synthesis of GSH. GSH is a tripeptide composed of γ-glutamate, cysteine and glycine. The sulfhydryl side chains of the cysteine residues of two glutathione molecules form a disulfide bond (GSSG) during the course of being oxidized in reactions with various oxides and peroxides in cells. Reduction of GSSG to two moles of GSH is the function of glutathione reductase, an enzyme that requires coupled oxidation of NADPH.
The cysteine thiol of GSH plays the role in reducing oxidized thiols in other proteins. Oxidation of 2 cysteine thiols forms a disulfide bond. Although this bond plays a very important role in protein structure and function, inappropriately introduced disulfides can be detrimental. Glutathione can reduce disulfides nonenzymatically. Oxidative stress also generates peroxides that in turn can be reduced by glutathione to generate water and an alcohol, or 2 waters if the peroxide were hydrogen peroxide.
Regeneration of reduced glutathione is carried out by the enzyme, glutathione reductase. This enzyme requires the co-factor NADPH when operating in the direction of glutathione reduction which is the thermodynamically favored direction of the reaction.
It should be clear that any disruption in the level of NADPH may have a profound effect upon a cells ability to deal with oxidative stress. No other cell than the erythrocyte is exposed to greater oxidizing conditions. After all it is the oxygen carrier of the body.