BCHEM 253 METABOLISM IN HEALTH AND DISEASES 1 Lecture 6 The Energetics of the Citric Acid Cycle Christopher Larbie, PhD

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1 BCHEM 253 METABOLISM IN HEALTH AND DISEASES 1 Lecture 6 The Energetics of the Citric Acid Cycle Christopher Larbie, PhD Introduction The 2 ATP s produced during glycolysis are only a small fraction of the potential energy available from glucose. Under anaerobic conditions, animals convert glucose into 2 molecules of lactate. Much of the potential energy of the glucose molecule remains untapped. Under Aerobic conditions a much more dynamic pyruvate metabolism occurs. The 2 moles of NADH produced by glyceraldehyde-3-phosphate dehydrogenase are oxidized in the electron transport chain back to NAD +. The electron transport chain generates a proton gradient that drives the synthesis of 5 ATP molecules from ADP and Pi. Furthermore, the pyruvate formed by glycolysis is converted to acetyl-coa by pyruvate dehydrogenase (generating another 2 moles of NADH per glucose and another 5 ATPs by oxidative phosphorylation). The acetyl-coa formed enters into the citric acid cycle where it is completely oxidized into CO 2. The electrons liberated by oxidation are captured by NAD + or FAD which are then transferred via the electron transport chain ultimately to O2, the final electron acceptor. The electron transport chain is coupled to generating a proton gradient which produces a proton motive force that drives the synthesis of ATP. This allows the net production of 32 molecules of ATP to be formed per glucose molecule. The function of the citric acid cycle is to harvest high energy electrons from carbon fuels. The citric acid cycle is the central metabolic hub of the cell, the gateway of aerobic metabolism. The citric acid cycle produces intermediates which are precursors for fatty acids, amino acids, nucleotide bases, cholesterol and porphyrins. The citric acid cycle is shown below. The citric acid cycle occurs in the mitochondria of eukaryotes. 1 P a g e D r. C h r i s t o p h e r L a r b i e

2 New carbons enter the citric acid cycle through acetyl-coa. The acetyl group may come from pyruvate, fatty acids, ketone bodies, ethanol or alanine. The two carbons of acetyl- CoA are transferred to oxaloacetate to yield the first tricarboxylic acid citrate in a reaction catalyzed by citrate synthase. A dehydration followed by a rehydration rearranges citrate into isocitrate. Two successive decarboxylations coupled to the generation of 2 NADH produce succinyl-coa. Four steps later, oxaloacetate is regenerated along with a GTP, FADH2 and NADH. The citric acid cycle may seem like an elaborate way to oxidize acetate into carbon dioxide, but there is chemical logic to the cycle. In order to directly oxidize acetate into two molecules of CO2, a C C bond must be broken. Under the mild conditions found in cells, there is insufficient energy to break the bond. Biological systems often break C C bonds between carbon atoms α and β to a carbonyl group. Examples are aldolase and transaldolase. Another common type of C C cleavage is α-cleavage of an α-hydroxy-ketone which we have seen before in transketolase and pyruvate decarboxylase, two TPP dependent enzymes. 2 P a g e D r. C h r i s t o p h e r L a r b i e

3 Neither of these common strategies for cleavage of C C bonds is available to acetate. Instead the acetate is activated in the form acetyl-coa, condensed with oxaloacetate to form citrate and then carrying out a β-cleavage in subsequent steps. The Net reaction of the citric acid cycle is: 3NAD + + FAD + GDP + Pi + Acetyl-CoA + 2H2O 3NADH + FADH2 + GTP + CoA + 2CO2 + 3H + The Aerobic Fate of Pyruvate In eukaryotes, the reactions of the citric acid cycle occur in the mitochondria. The mitochondrion is enclosed by a double membrane. All of the glycolytic enzymes are found in the cytosol of the cell. Charged molecules such as pyruvate must be transported in and out of the mitochondria. Small charged molecules with molecular weights of less than 10,000 can freely diffuse through the outer mitochondrial membrane through aqueous channels called porins. A transport protein called pyruvate translocase specifically transports pyruvate through the inner mitochondrial membrane into the matrix of the mitochondrion. 3 P a g e D r. C h r i s t o p h e r L a r b i e

4 Once in the mitochondria, pyruvate dehydrogenase converts pyruvate into acetyl-coa and CO2. This reaction may look straight forward but it is complex. Pyruvate dehydrogenase is a humongous enzyme that can be visualized by electron micrographs. Pyruvate dehydrogenase is a multienzyme complex held together by noncovalent interactions. There are three different enzymes and five coenzymes. The three individual enzymes are E1, pyruvate dehydrogenase, E2, dihydrolipoyl transacetylase, and E3, dihyrolipoyl dehydrogenase. The multienzyme complex consists of 24 subunits of E1, 24 subunits of E2 and 12 subunits of E3. In E1 we find a thiamine pyrophosphate coenzyme. In E2 there is a lipoic acid coenzyme. In E3 there is a FAD coenzyme. The molecular weight of pyruvate dehydrogenase isolated from E. coli is 4.6 million Daltons, slightly larger than a ribosome. In mammals, pyruvate dehydrogenase is twice that size and has two additional regulatory subunits. One is a protein kinase which phosphorylates three serine residues in E1; the other is a phosphatase which hydrolytically removes the same phosphoryl groups from the serines of E1. The structure of the pyruvate dehydrogenase complex is shown below. The E2, dihydrolipoyl transacetylase are shown as red balls. These 24 subunits create the core upon which the pyruvate dehydrogenase complex is built. The E1, pyruvate dehydrogenase enzyme is an α2β2 dimer shown as the orange and yellow balls. There are 24 of the αβ dimers in the complex. There are 12 subunits of the E3, dihyrolipoyl dehydrogenase complex shown in green. The E3 subunits associate into dimers, so there are six E3 dimers in the complex. 4 P a g e D r. C h r i s t o p h e r L a r b i e

5 Lets look at the chemistry going on in this complex. In E1, pyruvate dehydrogenase enzyme, we have a tightly bound thiamine pyrophosphate coenzyme or prosthetic group. We have encountered this cofactor before, pyruvate decarboxylase and transketolase. The reaction catalyzed in this subunit is similar to that of pyruvate decarboxylase. 5 P a g e D r. C h r i s t o p h e r L a r b i e

6 The next step in the enzyme catalyzed reaction involves a new cofactor we haven t encountered yet. It is lipoic acid. Lipoic acid is not found free in nature. Instead lipoic acid is covalently attached to a lysine residue through an amide linkage. The enzyme that catalyzes the formation of the lipoamide linkage uses ATP to generate AMP and pyrophosphate. Lipoic acid is an important cofactor because it couples acyl-group transfers with electron transfers during the oxidation and decarboxylation of α-keto acids. Because the ring strain inherent in the disulphide cyclic structure is relieved upon reduction, lipoic acid has a strong negative reduction potential, E o = 0.30 V. 6 P a g e D r. C h r i s t o p h e r L a r b i e

7 To summarize the chemistry up to this point, this enzyme has taken pyruvate, used TPP to catalyze the decarboxylation of an α-ketoacid to form the hydroxyethyl-tpp intermediate which is stable enough to isolate. The hydroxyethyl-tpp was then oxidized by and concomitantly transferred to lipoamide to form an acetyllipoamide. The acetyllipoamide is the final product of E1. The lipoamide prosthetic group is part of the E2 Dihydrolipoyl transacetylase enzyme. The next step in the course of the reaction mechanism occurs in the active site of the E2, Dihydrolipoyl transacetylase enzyme, which catalyzes the transfer of the acetyl group from lipoamide to coenzyme A. If you look back to the structure of lipoamide, you will see it has a long flexible tether linking the amide to the E2 enzyme. The length of this tether is about 45 Å. This is long enough to reach the active site of E1 to form the acetyllipoamide and then carry this intermediate to the active site of the E2 enzyme to form acetyl-coa and the dihydrolipoamide which is then carried to yet the third active site, the E3, dihyrolipoamide dehydrogenase enzyme. The E3, dihyrolipoamide dehydrogenase enzyme regenerates lipoamide for another round of catalysis. This enzyme is a flavoprotein, meaning, it has a tightly bound FAD prosthetic group. Flavins undergo 2 electron reductions, but can accept electrons one at a time, unlike NAD + which can only accept two electrons at a time in the form of a hydride. This gives flavoproteins more catalytic diversity than NAD coenzymes. 7 P a g e D r. C h r i s t o p h e r L a r b i e

8 The final step of the reaction catalyzed by this E3 enzyme is the transfer of the 2 electrons from the tightly bound FADH2 to the transiently bound NAD + to generate NADH + H +. This is a usual electron transfer. The common role of FAD is to accept electrons from NADH. One thing about flavoproteins is that the protein bound flavins have a great variety of reduction potentials. In this enzyme the reduction potential is shifted such that FADH2 is the electron donor and NAD + is the acceptor. Typical flavoprotein FAD + 2e - + 2H + FADH2 NAD + 2e - + H + NADH E o 0 V E o = V 8 P a g e D r. C h r i s t o p h e r L a r b i e

9 THE CYCLE I. Citrate Synthase 9 P a g e D r. C h r i s t o p h e r L a r b i e

10 The first reaction of the citric acid cycle is the condensation of acetyl-coa and oxaloacetate to form citrate and CoA-SH. The enzyme that catalyzes this reaction is called citrate synthase. (ΔG o = kj/mol) The change in free energy based on the steady state concentrations of oxaloacetate, acetyl-coa and citrate in the mitochondria isolated from pig hearts is: ΔG = kj/mol (a very exergonic reaction and irreversible). The mechanism of citrate synthase is shown below. In the active site of the enzyme there are two histidines and an aspartate which function as general acids and bases during catalysis. 10 P a g e D r. C h r i s t o p h e r L a r b i e

11 The first step is to generate an enol of acetyl-coa. Asp-375 functions as a general base abstracting a proton from the methyl group of Acetyl-CoA. His-274 concertedly functions as a general acid donating a proton to the carbonyl to form the enol. The enol generated in the first step is converted into a nucleophile by the abstraction of the enol hydrogen by His- 274 now functioning as a general base. The electrons of the double bond attack the electrophilic center of the ketone of oxaloactete. His-320 functions in concert as a general acid donating its proton to the carbonyl oxygen of oxaloacetate to form citryl-coa. Citryl-CoA spontaneously hydrolyses while it is still bound to the active site to generate coenzyme A and citrate. Citrate synthase is a homodimer with symmetry as shown below. It has a sequential order kinetic mechanism. First the enzyme binds oxaloacetate which induces the large conformational change shown (b). This is yet another example of induced fit. The conformation change induced by oxaloacetate binding creates the acetyl-coa binding site and seals oxaloacetate form the aqueous solvent. Citrate synthase is allosterically regulated by NADH and Succinyl- CoA. It is the first step of a metabolic pathway. It catalyzes an irreversible step in the pathway. It is a homodimer with symmetry. 11 P a g e D r. C h r i s t o p h e r L a r b i e

12 II. Aconitase Citrate is a tertiary alcohol. It is difficult to oxidize tertiary alcohols because forming the ketone would involve breaking a carbon-carbon bond. To get around this problem, citrate is isomerized into isocitrate. Isocitrate is a secondary alcohol which can be easily oxidized to the ketone by NAD +. The enzyme that catalyzes this migration of a hydroxyl group is aconitase. This enzyme catalyzes the dehydration of citrate of form cis-aconitate and then rehydrating the double bond to form isocitrate. (ΔG o = +6.7 kj/mol) The change in free energy based on the steady state concentrations of citrate and isocitrate in the mitochondria isolated from pig hearts is: ΔG = +0.8 kj/mol (near equilibrium). Aconitase is an iron sulphur protein (shown above). It contains four iron atoms complexed to four inorganic sulphides and three cysteine sulphur atoms called a 4Fe-4S iron- sulphur cluster. One of the iron atoms has an open coordination site that complexes with the carboxylate group of C3 and the hydroxyl group of citrate. This iron residue facilitates the dehydration and rehydration reaction and accounts for the stereospecifity of the reaction. 12 P a g e D r. C h r i s t o p h e r L a r b i e

13 III. Isocitrate Dehydrogenase This is the first oxidation step of the pathway. Isocitrate dehydrogenase catalyzes the oxidation of isocitrate to oxalosuccinate which is a β keto acid. This β keto acid spontaneously decarboxylates to form α ketoglutarate. ΔG o = -8.4 kj/mol. The change in free energy based on the steady state concentrations of isocitrate and α ketoglutarate in the mitochondria isolated from pig hearts is: ΔG = kj/mol (A very exergonic, irreversible reaction). This irreversible reaction is allosterically regulated. NADH and ATP are allosteric inhibitors. ADP is an allosteric activator which lowers the Km for isocitrate by a factor of 10. When the concentration of ADP is low, the Km for citrate is well above the physiological concentration making the enzyme essentially active. With ADP bound in the allosteric binding site, the Km is lowered by a factor of ten making the enzyme active at physiological concentrations. IV. α Ketoglutarate Dehydrogenase The next step in the TCA cycle is the oxidative decarboxylation of α ketoglutarate to form succinyl-coa and NADH. ΔG o = kj/mol The change in free energy based on the steady state concentrations of α ketoglutarate and succinyl-coa in the mitochondria isolated from pig hearts is: ΔG = kj/mol (a very exergonic, irreversible reaction). 13 P a g e D r. C h r i s t o p h e r L a r b i e

14 If you look at the chemical transformation involved, you will note the similarity between this enzymatic reaction and the one catalyzed by pyruvate dehydrogenase. Pryruvate dehydrogenase: Pyruvate + CoA + NAD + acetyl-coa + CO2 + NADH α Ketoglutarate dehydrogenase: α ketoglutarate + CoA + NAD + succinyl-coa + CO2 + NADH Both of these reactions include the decarboxylation of an α-keto acid and the subsequent formation of an high energy thioester linkage with coenzyme A. α Ketoglutarate dehydrogenase is a humongous multienzyme complex that is very similar to the pyruvate dehydrogenase multienzyme complex. The mechanism for α Ketoglutarate dehydrogenase is identical to that of pyruvate dehydrogenase. V. Succinyl-CoA Synthetase So far we have generated 2 molecules of CO2 and 2 molecules of NADH for each molecule of pyruvate that enters the TCA cycle. The electrons of NADH will be routed through the electron transport chain and ultimately generated 2.5 equivalents of ATP by oxidative phosphorylation. The succinyl-coa contains a high energy bond which is going to be utilized in this step of the cycle. 14 P a g e D r. C h r i s t o p h e r L a r b i e

15 The enzyme succinyl-coa synthetase couples the conversion of succinyl-coa into succinate with the synthesis of GTP from GDP and Pi. The standard free energy change for hydrolyzing succinyl-coa into succinate and coenzyme A is kj/mole. The standard free energy required to synthesize GTP from GDP and Pi is kj/mole. If we couple these two reactions together than the standard free energy change is -3.3 kj/mole. This enzyme catalyzes a substrate level phosphorylation to generate the only NTP produced directly in the citric acid cycle. The GTP produced is converted into ATP by the enzymatic activity of nucleoside diphosphate kinase: ADP + GTP ATP + GDP For succinyl-coa synthetase: ΔG o = -3.3 kj/mol. The change in free energy based on the steady state concentrations of all the reactants and products in the mitochondria isolated from pig hearts is: ΔG 0 (a near equilibrium reaction). The mechanism for this enzyme is shown below. 15 P a g e D r. C h r i s t o p h e r L a r b i e

16 In the first step an enzyme bound inorganic phosphate group attacks the thioester to convert the high energy thioester bond into a high energy acylphosphate bond. This enzyme bound succinyl-phosphate then undergoes nucleophilic attack by an active site histidine residue to form succinate and a phosphorylated histidine intermediate. In the last step of this mechanism, GDP is bound and the phosphate group is transferred to GDP forming GTP. The potential energy of the thioester bond has been conserved first by the formation of succinyl-phosphate, then by the formation of the phosphorylated histidine residue and finally by the substrate level phosphorylation of GDP to form GTP. 16 P a g e D r. C h r i s t o p h e r L a r b i e

17 VI. Succinate Dehydrogenase In the next step succinate dehydrogenase is going to oxidize succinate into fumarate. The oxidation of an alkane is not sufficiently exergonic to reduce NAD + E o = The oxidation of an alkane is sufficiently exergonic to reduce FAD. E o = Fumarate + 2e - +2H + Succinate E o = ΔE o = V= ΔG o = kj/mol The change in free energy based on the steady state concentrations of all the reactants and products in the mitochondria isolated from pig hearts is: ΔG 0; (a near equilibrium reaction). Succinate dehydrogenase is an integral membrane bound enzyme that is part of the electron transport chain. All of the other enzymes of the citric acid cycle are soluble proteins found in the mitochondrial matrix. Succinate dehydrogenase also contains three iron sulphur clusters as part of its electron transport chain. The electrons captured by the FAD are passed to the iron sulphur clusters to coenzyme Q (quinine) to produce the reduced form QH2 (dihydroquinone). 17 P a g e D r. C h r i s t o p h e r L a r b i e

18 VII. Fumarase Fumarase catalyzes the trans addition of water to the double bond of fumarate to produce malate. The reaction catalyzed by fumarase is stereospecific. (ΔG o = -3.8 kj/mol). The change in free energy based on the steady state concentrations of all the reactants and products in the mitochondria isolated from pig hearts is: ΔG 0 (A near equilibrium reaction). VIII. Malate Dehydrogenase The last step is catalyzed by malate dehydrogenase. Malate dehydrogenase catalyzes the oxidation of the hydroxyl group of malate into a ketone to generate oxaloacetate and complete the journey around the cycle. ΔG o = kj/mol. The change in free energy based on the steady state concentrations of malate and oxaloacetate in the mitochondria isolated from pig hearts is: ΔG 0 (a near equilibrium reaction). Note that the standard change in free energy is large and endergonic. In order to drive this reaction the concentration of oxaloacetate must be maintained at a very low concentration (less than 10-6 M) in the mitochondrial matrix. IX The Net reaction of the citric acid cycle is: 3NAD + + FAD + GDP + Pi + Acetyl-CoA + 2H2O 3NADH + FADH2 + GTP + CoA + 2CO2 + 3H + ΔG o = - 40 kj/mol The change in free energy based on the steady state concentrations of all the reactants and products in the mitochondria isolated from pig hearts is: ΔG = -115 kj/mol 18 P a g e D r. C h r i s t o p h e r L a r b i e

19 REGULATION OF THE CITRIC ACID CYCLE I. Changes in Free Energy 3 Molecules of NADH and 1 molecule of FADH2 are generated each turn of the Citric acid cycle. The eight electrons captured are transported by electron carriers to O 2 generating a proton gradient that drives the oxidative phosphorylation of ADP to generate ATP. The stoichiometry of electron transport and oxidative phosphorylation is 2.5 ATP per NADH and 1.5 ATP per FADH2. As a result 9 ATP are generated by electron transport-oxidative phosphorylation per turn of the cycle. Molecular oxygen does not participate in the citric acid cycle. However, the cycle operates only under aerobic conditions because NAD + and FAD cannot be regenerated in the absence of oxygen. Glycolysis has an aerobic and anaerobic mode, but the citric acid cycle only operates under aerobic conditions. II. Regulation of Pyruvate Dehydrogenase Pyruvate is an important metabolite. It is the product of glycolysis and a substrate for gluconeogenesis. Under anaerobic conditions, pyruvate is fermented into lactate or alcohol to regenerate NAD +. Under aerobic conditions pyruvate is converted into acetyl-coa by pyruvate dehydrogenase. This is an irreversible step in animals. Animals are unable to directly convert acetyl-coa back to pyruvate. The oxidative decarboxylation of pyruvate to form acetyl-coa commits the acetyl group to either complete oxidation by the citric acid cycle into CO2 or incorporation in fatty acids and lipids. This irreversible branch point is regulated by several means. This enzyme is allosterically regulated. High concentrations of acetyl-coa inhibit the transacetylase 19 P a g e D r. C h r i s t o p h e r L a r b i e

20 component of E2. High concentrations of NADH inhibits the dihydrolipoyl dehydrogenase component of E3. In Mammals, pyruvate dehydrogenase is regulated by phosphorylation by pyruvate dehydrogenase kinase a regulatory enzyme that is part of the mammalian pyruvate dehydrogenase complex. Pyruvate dehydrogenase kinase is allosterically activated by NADH and acetyl-coa such that when the mitochondrial matrix concentrations of these two effectors rises, they stimulate the phosphorylation of a serine residue on the pyruvate dehydrogenase E1 subunit, which blocks the first step of catalysis, the decarboxylation of pyruvate. Pyruvate allosterically inhibits the kinase s activity. Eventually the mitochondrial matrix concentrations of NADH and acetyl-coa will come down and the enzyme will need to be reactivated. The reactivation of this enzyme is accomplished by another enzyme, pyruvate dehydrogenase phosphatase, a Ca 2+ activated enzyme that binds to the dehydrogenase complex, hydrolyses the 20 P a g e D r. C h r i s t o p h e r L a r b i e

21 phosphorylated serine, reactivating the enzyme. This phosphatase will remain associated with the dehydrogenase complex as long as the NADH/NAD + and the acetyl-coa/coa ratios remain low. High concentrations of NADH or acetyl-coa inactivate the phosphatase and cause it to dissociate from the pyruvate dehyrdrogenase complex. Insulin and Ca 2+ ions activate the phosphatase. Important to note that the camp dependent phosphorylation cascade has no effect on the phosphorylation of pyruvate kinase. The cascade produced by insulin however does activate the phosphatase. III. Regulation of the Citric Acid Cycle The citric acid cycle must be carefully regulated by the cell. If the citric acid cycle were permitted to run unchecked, large amounts of metabolic energy would be wasted in the over production of reduced coenzymes and ATP. Conversely if the citric acid cycle ran too slowly, ATP would not be generated fast enough to sustain the cell. By looking at the changes in free energy of the reactions of the citric acid cycle, it is clear that there are three irreversible steps. These three reactions of the cycle, citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase operate with large negative free energy changes under the concentrations of products and reactants in the matrix of the mitochondria. Because the citric acid cycle is linked to oxygen consumption to regenerate NAD +, the citric acid cycle is regulated primarily by product feedback inhibition. Glycolysis and glycogen metabolism are under complex systems of allosteric and hormonal control. The citric acid cycle in contrast is regulated by three simple mechanisms. 1. Substrate availability 2. Product inhibition 3. Competitive feedback inhibition. The allosteric effectors that control the flux of metabolites through the citric acid cycle are shown on the next page. The principle signals are acetyl-coa, succinyl-coa, ATP, ADP, AMP, NAD + and NADH. All of the regulatory enzymes of the citric acid cycle including pyruvate dehyrogenase are allosterically inhibited by NADH. The combination of the electron transport chain and oxidative phosphorylation produce ATP form NADH, consequently ATP is an allosteric inhibitor of pyruvate dehydrogenase and isocitrate dehydrogenase. The TCA cycle is turned on however by high ratios of either ADP/ATP or NAD + /NADH which indicate that the cell has run low of NADH or ATP. 21 P a g e D r. C h r i s t o p h e r L a r b i e

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23 IV. Citric Acid Cycle Intermediates Are Precursors for Biosynthetic Reactions It is easy to think of the citric acid cycle as a catabolic pathway oxidizing acetate into CO2 and generating ATP. Many of the intermediates, α-ketoglutarate, succinyl-coa, fumarate and oxaloacetate produced in the citric acid cycle, are precursors for important biomolecules. 1. A Transamination reaction converts glutamate into α-ketoglutarate or vice versa. Glutamate is a precursor for the synthesis of other amino acids and purine nucleotides. 2. Succinyl-CoA is a precursor for porphyrins. 3. Oxaloacetate can be transaminated to form aspartate. Aspartate itself is a precursor for other amino acids and pyrimidine nucleotides. Oxaloacetate is a substrate for gluconeogenesis. 4. Citrate can be exported out of the mitochondria into the cytostol where is broken down by ATP citrate lyase to yield oxaloacetate and acetyl-coa. (Citrate Oxaloacetate shuttle shown below) 5. The acetyl-coa produced is a precursor for fatty acids. The oxaloacetate produced in this reaction is reduced to malate which can be either be transported back into the matrix of the mitochondria where it is reoxidized into oxaloacetate or malate can be oxidatively decarboxylated to pyruvate by malic enzyme which can then be transported back to the mitochondria. 23 P a g e D r. C h r i s t o p h e r L a r b i e

24 V. Anaplerotic Reactions Reciprocal arrangements replenish the intermediates removed from the citric acid cycle for biosynthesis. The reactions that replenish the citric acid cycle are called anaplerotic reactions which means filling up reactions. Anaplerotic reactions include PEP carboxylase and pyruvate carboxylase both of which synthesize oxaloacetate from pyruvate. Pyruvate carboxylase is one of the most important anaplerotic reactions. This enzyme catalysis the first step of gluconeogenesis (synthesis of glucose from non-carbohydrate precursors) from pyruvate and is found exclusively in the mitochondria. The enzyme has a covalently bound biotin cofactor. Since this enzyme functions in gluconeogenesis, it is allosterically regulated. This enzyme requires acetyl-coa to be bound at an allosteric binding site in order to activate bicarbonate with ATP. PEP carboxylase is found in yeast, bacteria and plants but not in animals. 24 P a g e D r. C h r i s t o p h e r L a r b i e

25 Malic enzyme catalyses the reaction shown above. The malic enzyme is found in the cytosol or mitochondria of many plants and animals and is a NADP + dependent enzyme. Other metabolites fed into the citric acid cycle are succinyl-coa from the degradation of odd-chain fatty acids, α-ketoglutarate and oxaloacetate from the transamination reactions of glutamate and aspartate. VI. The Glyoxylate Cycle of Plants, Yeast and Bacteria Plants, fungi, algae, protozoans and bacteria can thrive on two carbon compounds such as acetate, ethanol and acetyl-coa, as their sole carbon source. In the citric acid cycle, we have seen how acetyl-coa is oxidized into 2 molecules of CO2 to generate ATP. There is no net synthesis in the TCA cycle since we end up with oxaloacetate, the exact same compound we began with. The TCA cycle has evolved to produce ATP from acetyl-coa. This cycle cannot produce massive amounts of biosynthetic precursors needed for growth on acetate unless alternative reactions are available. In order to grow and thrive on acetate, the two CO2 producing reactions of the citric acid cycle need to be bypassed. The living organisms listed above can grow on acetate by employing a modification of the citric acid cycle called the glyoxylate cycle which takes two carbon compounds and converts them into four carbon compounds. This cycle bypasses the oxidative decarboxylations of the citric acid cycle by using two alternative enzymes, Isocitrate lyase and malate synthase. Isocitrate lyase cleaves isocitrate into glyoxylate and succinate. Malate synthase takes glyoxylate and condenses it with another acetyl-coa to form Malate and CoA. The net effect is the preservation of carbon units using two molecules of acetyl-coa to generate malate which is then converted by malate dehydrogenase into oxaloacetate which can be then converted into PEP and on through gluconeogenesis. The enzymes of the glyoxylate 25 P a g e D r. C h r i s t o p h e r L a r b i e

26 cycle are contained in glyoxysomes which are organelles devoted to this cycle. There are enzymes common to both the TCA and glyoxylate cycles as well as isozymes and functionally distinct enzymes allowing these two organelles to operate independently in these two cycles. The glyoxylate cycle allows seeds to grow in the dark where photosynthesis is impossible. Many seeds are rich in lipids allowing these organisms to degrade fatty acids to generate acetyl-coa. The glyoxylate cycle then can take two of these acetyl- CoA s to generate oxaloacetate and from there other intermediates. The glyoxysomes lack succinate dehydrogenase, fumarase and malate dehydrogenase. Consequently intermediates must be shuttle back and forth between the glyoxysome and the mitochondria. Succinate is transported to the mitochondria where it is converted into oxaloacetate. The transamination of aspartate is necessary because oxaloacetate cannot be transported out of the mitochondria. Aspartate then moves from the mitochondria back to the glyoxysome where it is transaminated back to oxaloacetate and completes the shuttle. 26 P a g e D r. C h r i s t o p h e r L a r b i e

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