Metabolism and Energy metabolism. (Determination of pyruvic acid contents in urine).
Investigation of Krebs cycle functioning. (Determination of the muscles’ succinatedehydrogenase).
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
compounds. Autotrophs 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
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
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:
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,
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.
the molecular weight of glucose is 180g/m, the
cellular respiration produces
the MW of ATP is 507 g/m, that would be equivalent
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 × 1 hr/60 min × 1000 g/kg = about 27.8 g/min.
Since the molecular weight of
ATP is 507 g/m, then
that 27.8 g/min × 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 × 6.02 x 1023 molecules/mole = 3.30 x 1022 molecules/min.
or, since there are 60
sec/min, then that’s
3.30 x 1022 molecules/min × 1min/60 sec = 5.50 x 1020 molecules/sec made by a
so that would be equivalent to
5.50 x 1020 molecules/sec ÷
or × 1kg/1000 g = 7.85 × 1015 molecules/sec/g of body
or × 1g/1000 mg = 7.85 × 1012 molecules/sec/mg of body
or × 1mg/1000 µg = 7.85 × 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
4. As NADH delivers more H+ and electrons into
the ETS, the proton gradient increases, with H+ building up outside
the inner mitochondrial membrane, and
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 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 +
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.
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.
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.
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.
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].
The various intermediates are present at very different concentrations. Citrate, oxoglutarate, succinate and malate are major metabolites, and are typically present at concentrations approaching 1 mM in the matrix space. Isocitrate is about 5% of the citrate concentration, and fumarate about 25% of malate, fixed by enzyme equilibrium constants. Coenzyme A and its various derivatives are fifty times lower than this, and oxaloacetate is present in vanishingly small amounts, especially in the fasting state. Lack of oxaloacetate is a major constraint on citrate synthase activity.
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.
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+.
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.
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.
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.
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.
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:
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 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.
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.
The energy used by human cells requires the hydrolysis of 100 to 150 moles of ATP daily which is around 50 to 75 kg. Typically, a human will use up their body weight of ATP over the course of the day. This means that each ATP molecule is recycled 1000 to 1500 times during a single day (100 / 0.1 = 1000). ATP cannot be stored, hence its consumption being followed closely by its synthesis.
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:
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.
In 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 > ADO), purinergic nucleotides like ATP are not strong agonists of P1 receptors which are strongly activated by adenosine and other nucleosides (ADO > AMP > ADP > ATP). P1 receptors have A1, A2a, A2b, and A3 subtypes ("A" as a remnant of old nomenclature of adenosine receptor), all of which are G protein-coupled receptors, A1 and A3 being coupled to Gi, and A3 being coupled to Gs.
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.