PYRUVATE DEHYDROGENASE and the CITRIC ACID CYCLE (TRICARBOXYLIC ACID CYCLE / KREBS CYCLE)

PYRUVATE DEHYDROGENASE ENZYME COMPLEX - multienzyme complex - irreversible reaction glucose cannot be formed from acetyl CoA - major source of acetyl CoA A. Function - links glycolysis and TCA cycle B. Location - mitochondrial matrix C. Component Enzymes 1. Pyruvate dehydrogenase (E1, decarboxylase) 2. Dihydrolipoyl transacetylase (E2) 3. Dihydrolipoyl dehydrogenase (E3) - also contains tightly bound regulatory enzymes 1. Protein kinase 2. Phosphoprotein phosphatase D. Coenzymes - act as carriers or oxidants for the intermediates of the reaction a. E1 - requires thiamine pyrophosphate b. E2 - requires - lipoic acid - CoA c. E3 - requires - FAD - NAD+ 1. Thiamine or Niacin Deficiency inactive pyruvate dehydrogenase no sufficient ATP production via the TCA cycle brain cell dysfunction E. Reactions

1. Pyruvate Decarboxylase Activity (Step 1) - catalyzed by the E1 subunit of PDH - thiamine pyrophosphate (TPP) as cofactor - CO2 is formed - the -hydroxyethyl group derived from pyruvate becomes covalently bound to TPP 2. Transfer of 2-Carbon Unit from E1 to E2 (Step 2) - E2 requires coenzyme lipoic acid (covalently attached to a lysine residue of the protein) - lipoamide (covalently bound form of lipoic acid) - hydroxyethyl group is oxidized to an acetyl group transferred to the oxidized form of lipoamide - TPP is regenerated at this step

3. Dihydrolipoyl Transacetylase Activity (Step 3) - catalyzed by E2 subunit of PDH - transfer of acetyl group from lipoamide to CoA acetyl CoA - lipoamide is in the reduced state after the transfer 4. Dihydrolipoyl Dehydrogenase Activity (Step 4) - catalyzed by E3 subunit of PDH - cofactors - NAD+ - FAD (reoxidizes lipoamide and is reduced to FADH2) 5. FADH2 Reoxidation to FAD by NAD+ - products - NADH - H+

F. PDH Regulation 1. Product Inhibition - acetyl CoA - NADH 2. Substrate Availability - CoA - NAD+

3. Covalent Modification a. 2 Forms of PDH i. Inactive - phosphorylated ii. Active - dephosphorylated b. Protein Kinase - cyclic AMP-independent - phosphorylates a serine residue on the decarboxylase (-subunit) of PDH inactivation of E1 i. Dependent on - Mg++ - ATP

ii. Regulation iia. Stimulated by - ATP - acetyl CoA - NADH - high-energy signals pyruvate dehydrogenase complex turned off iib. Inhibited by - CoA - NAD+ - pyruvate - low-energy signals pyruvate dehydrogenase complex turned on - elevated pyruvate levels protein kinase inhibited E1 maximally active c. Phosphoprotein Phosphatase - reactivate the complex (E1) i. Regulation - activated by increasing [Ca++] in the mitochondria (occurs when the system is short of ATP) ii. Calcium - strong activator - stimulates E1 activity - skeletal muscle contraction Ca++ released pyruvate dehydrogenase complex activation energy production d. Insulin - activate PDH in adipose tissue G. Genetic Defects in PDH - PDH Deficiency - most common biochemical cause of congenital lactic acidosis 1. Results - decreased or complete loss of enzyme activity inability to convert pyruvate to acetyl CoA pyruvate shunted to lactic acid via lactate dehydrogenase a. Brain - relies on the TCA cycle for most of its energy - sensitive to acidosis 2. Symptoms - lactic acidosis - neurologic disorders a. Milder Cases - causes episodic ataxia (inability to coordinate voluntary muscles) - induced by carbohydrate-rich meal i. E1 Defect - affects both males and females X-linked dominant b. Moderate Cases - moderate lactic acidosis - profound psychomotor retardation - damage to the - cerebral cortex - basal ganglia - brain stem - death in infancy c. Severe Cases overwhelming lactic acidosis usually fatal (neonatal death) 3. Treatments a. Thiamine - large doses - may be effective for defects in E1 (reduces affinity of the enzyme for TPP) b. Lipoic Acid - large doses - may be effective for defects in E2

c. Ketogenic Diet a. High Fat - alternate fuel supply in the form of ketone bodies b. Low Carbohydrate lowers pyruvate and lactate (formed from the excess pyruvate) levels H. Case: PDH Deficiency 1. Presentation A male infant was born without complications and was considered normal until 13 months of age when he had trouble standing. At 20 months of age he was admitted to the hospital, where he exhibited severe ataxia and psychomotoric retardation. He could not stand or walk, and he spoke very little. No abnormal neurologic signs were observed. Urinalysis revealed high level of alanine. Blood analysis revealed high levels of pyruvate and lactate. 2. Diagnosis and Treatment A minor deficiency of PDH was suspected. Therapeutic doses of thiamine (600 mg 3x/day) were administered and within 6 months the boy could walk and speak. The symptoms recurred when thiamine therapy was discontinued but again were controlled when therapy was reinstated. A follow-up examination at 3 years indicated that the childs development had returned to normal and that serum pyruvate and lactate levels were within normal ranges. 3. Discussion In this patient, high serum pyruvate levels suggested PDH deficiency. Lactate and alanine levels were also high, since they may be formed from pyruvate by the reversible activities of LDH and alanine transaminase. The patients defect is apparently in the E1 subunit of PDH, which causes a reduced affinity for TPP. This reduced affinity can be overcome by increasing the concentration of TPP, which is achieved by increasing thiamine intake, the precursor of TPP. I. Arsenic Poisoning inhibition of enzymes that require lipoic acid as cofactor pyruvate and lactate accumulation brain effects neurologic disturbances and death - pyruvate dehydrogenase - -ketoglutarate dehydrogenase - branched-chain -keto acid dehydrogenase 1. Arsenite - trivalent form of arsenic - forms a stable complex with the thiol groups (-SH) of lipoic acid OVERVIEW of the TCA CYCLE A. Description - series of enzymatically catalyzed reactions forming common pathway for the oxidation of all metabolic fuels (carbohydrates, free fatty acids, ketone bodies, amino acids) (oxidation of acetyl CoA to CO2 and H2O) - occurs totally in the mitochondrial matrix (in close proximity to the electron transport reactions) - O2 as the final electron acceptor B. Functions 1. An Amphibolic Pathway - involved in both anabolic and catabolic processes a. Anabolic Reactions - intermediates are used in the biosynthesis of many compounds b. Catabolic Reactions - provides a means for the degradation of 2-carbon acetyl residues derived from carbohydrates, free fatty acids and amino acids 2. Provides much of the Energy for Respiration - electrons generated in the cycle electron transport chain oxidative phosphorylation ATP synthesis (accounts for about 2/3 of the total oxygen consumption and ATP production)

C. Stoichiometry of the TCA Cycle Acetyl CoA + 3NAD+ + FAD + GDP + Pi + 2H2O 2CO2 + 3NADH + FADH2 + GTP + 3H+ + CoA 1. Summary of Reactions - 2 carbon atoms (acetyl CoA) TCA cycle - no net production or consumption of any cycle intermediate - 4 pairs of electrons are transferred during 1 turn of the cycle a. 3 pairs of electrons reducing NAD+ NADH b. 1 pair of electrons reducing FAD FADH2

2. ATP Yield - oxidation of 1 NADH by the electron transport chain 3 ATP - oxidation of 1 FADH2 by the electron transport chain 2 ATP

REACTIONS of the TCA CYCLE A. Synthesis of Citrate from Acetyl CoA and OAA - aldol condensation (condensation reaction) catalyzed by citrate synthase - uses an intermediate of the TCA cycle produces an intermediate of the TCA cycle entry of acetyl CoA to the TCA cycle does not lead to net production or consumption of cycle intermediates 1. Inhibitors - ATP - NADH - succinyl CoA - acyl CoA derivatives of fatty acids 2. Activators - Ca++ - ADP 3. Primary Mode of Regulation - availability of substrates - acetyl CoA - oxaloacetate 4. Other Functions of Citrate - source of acetyl CoA for the cytosolic synthesis of fatty acids - inhibits PFK (rate-limiting enzyme of glycolysis) - activates acetyl CoA carboxylase (rate-limiting enzyme of fatty acid synthesis) B. Citrate Isomerization (to Isocitrate) - catalyzed by aconitase (has Fe-S center as prosthetic group) - inhibited by fluoroacetate - compound used as rat poison - converted to fluoroacetyl CoA condenses with oxaloacetate fluorocitrate (inhibitor of aconitase) citrate accumulation - interconversion of citrate and isocitrate requiring 2 steps 1. Dehydration of citrate cis-aconitate 2. Hydration of cis-aconitate isocitrate C. Oxidative Decarboxylation of Isocitrate (to -Ketoglutarate) - irreversible oxidative decarboxylation catalyzed by isocitrate dehydrogenase 1. Activator - ADP - Ca++ 2. Inhibitors - ATP - NADH - yields the NADH and releases CO2 - 1 of the rate-limiting steps of the cycle

D. Oxidative Decarboxylation of -Ketoglutarate (to Succinyl CoA) - catalyzed by -ketoglutarate dehydrogenase complex (3 enzymatic activities) - yields the NADH and releases CO2 - not regulated by phosphorylation/dephosphorylation reactions 1. Coenzymes - thiamine pyrophosphate (TPP) - lipoic acid - FAD - NAD+ - CoA 2. Structure - multimeric protein - very similar to PDH (E3 subunit is similar to that of PDH) 3. Product - succinyl CoA - energy-rich thioester similar to acetyl CoA

4. Inhibitors - ATP - GTP - NADH - succinyl CoA 5. Activator - Ca++ 6. -Ketoglutarate - also produced by the oxidative deamination or transamination of glutamate E. Cleavage of Succinyl CoA (to Succinate) - catalyzed by succinate thiokinase (succinyl CoA synthetase) - cleaves high-energy thioester bond of succinyl CoA - coupled to phosphorylation of GDP GTP - GTP - energy content is the same as of ATP - interconverted with ATP by nucleoside diphosphate kinase reaction GTP + ADP GDP + ATP 1. Substrate Level Phosphorylation - production of high energy phosphate coupled to the conversion of a substrate product 2. Source of Succinyl CoA - from fatty acids with odd number of carbon atoms - from propionyl CoA derived from the metabolism of branched-chain amino acids 3. Fate of Succinyl CoA - biosynthesis of heme F. Oxidation of Succinate (to Fumarate) 1. Enzyme - succinate dehydrogenase a. Prosthetic Groups - 3 different types of Fe-S centers - covalently bound FAD b. Association - tightly associated with the inner mitochondrial membrane c. Inhibitor - oxaloacetate 2. Reaction Description - dehydrogenation reaction - FAD reduction FADH2 - reducing power of succinate not sufficient to reduce NAD + FAD as electron acceptor 3. Malonate - dicarboxylic acid - structural analogue of succinate - competitively inhibits succinate dehydrogenase G. Hydration of Fumarate (to Malate) - freely reversible reaction - catalyzed by fumarase (fumarate hydratase) - hydration reaction (H2O is added to the carbon-carbon double bond) 1. Fumarate - also produced - by the urea cycle - in purine synthesis - catabolism of - phenylalanine - tyrosine

H. Oxidation of Malate (to Oxaloacetate) - catalyzed by malate dehydrogenase - dehydrogenation reaction - NAD+ reduction NADH 1. Oxaloacetate - also produced by the transamination of aspartic acid

The Citric Acid Cycle - major catabolic pathway for acetyl-CoA in aerobic organisms - nine ATP are generated via oxidative phosphorylation and one ATP (or GTP) arises at substrate level from the conversion of succinyl-CoA to succinate per cycle Acetyl-CoA - product of carbohydrate, protein, and lipid catabolism - taken into the cycle oxidized to CO2 release of reducing equivalents (2H) oxidation of 2H in the respiratory chain phosphorylation of ADP to ATP

VITAMINS in the TCA CYCLE - 4 of the soluble vitamins of the B complex A. Riboflavin - gives rise to FAD B. Niacin - in the form of NAD C. Thiamine (B1) - as thiamine diphosphate - coenzyme for decarboxylation in the -ketoglutarate dehydrogenase reaction D. Pantothenic Acid - as part of CoA ANAPLEROTIC REACTIONS - can increase the concentration of TCA cycle intermediates increased oxidation rate of 2-carbon units - intermediates may also be used for biosynthetic reactions must be replaced A. Sources of Carbon for Anaplerotic Reactions

Involvement of the Citric Acid Cycle in Transamination and Gluconeogenesis

1. Amino Acid Metabolism which produces a. Transaminases that form -ketoglutarate b. Transaminases that form OAA c. Glutamate Dehydrogenase - provides -ketoglutarate Glutamate + NAD+ + H2 -Ketoglutarate + NH3 + NADH + H+ d. Succinyl CoA Formation from - isoleucine - valine - methionine - threonine 2. Pyruvate Carboxylase - forms OAA

B. TCA in Fatty Acid Synthesis

REGULATION of the TCA CYCLE A. Regulation by Activation and Inhibition of Enzyme Activities 1. Regulated Enzymes a. Citrate synthase b. Isocitrate dehydrogenase c. -ketoglutarate dehydrogenase complex B. Regulation by ADP Availability 1. Effects of Elevated ADP - energy consumption (muscle contraction, biosynthetic reactions) ATP hydrolysis ADP + Pi increased ADP levels acceleration of reactions that use ADP ATP generation (most important is oxidative phosphorylation) ATP production increases until it matches the rate of ATP consumption by energy-requiring reactions 2. Effects of Low ADP a. Respiratory Control of Energy Production - limited ADP (phosphate acceptor) or Pi decreased ATP formation by oxidative phosphorylation (rate of oxidative phosphorylation is proportional to [ADP][Pi]/[ATP]) - oxidation of NADH and FADH2 by the respiratory chain also stops if ADP is limiting (processes of oxidation and phosphorylation are tightly coupled and must occur simultaneously) NADH and FADH2 accumulation NAD+ and FAD depletion inhibition of acetyl CoA oxidation by the TCA cycle (due to lack of oxidized coenzymes)

SUMMARY