Pyruvate Dehydrogenase and the TCA Cycle Biochem 4511 Figures Essential Biochemistry 3rd Ed Pratt and Cornely Principles of Biochemistry 5th Ed Moran et al Fundamentals of Biochemistry 2nd Ed Voet Voet and Pratt Citric acid cycle converts two carbon acetyl CoA into CO2 and therefore represents the final stage in the oxidation of metabolic fuels such as glucose It consists of eight reactions in a cyclic manner Pyruvate Dehydrogenase The pyruvate dehydrogenase complex includes three enzymes that collectively remove a carboxylate group from pyruvate and produce acetyl CoA and NADH Surface View Cutaway View Pyruvate Dehydrogenase Complex E1 pyruvate dehydrogenase E2 dihydrolipoamide acetyltransferase E3 dihydrolipoamide dehydrogenase Pyruvate Dehydrogenase Complex Pyruvate Dehydrogenase Complex E1 pyruvate dehydrogenase E2 dihydrolipoamide acetyltransferase E3 dihydrolipoamide dehydrogenase The pyruvate dehydrogenase complex is several times larger than a ribosome In eukaryotes there are 60 E1 60 E2 and 12 E3 enzymes in the complex a 132 subunit complex Co factors play a key role forming a small electron transfer chain e from pyruvate lipoamide FAD NAD Pyruvate Dehydrogenase reaction is complex Actual Reaction Pyruvate Dehydrogenase Activity There are 5 steps to pyruvate dehydrogenase activity 1 E1 decarboxylates pyruvate through a thiamine pyrophosphate TPP co factor CO2 is released 2 The remaining hydroxyethyl group is transferred to the E2 enzyme by a lipoamide co factor 3 The acetyl group is transferred to CoA through the formation of a thioester bond 4 Lipoamide of E2 is regenerated by oxidation through a FAD co factor reduced to FADH2 on the E3 enzyme 5 E3 is regenerated and NADH is produced which is the final product of the catalytic cycle Thiamine Vitamin B1 is a common co factor in decarboxylation reactions and transketolase in the pentose phosphate pathway Lipoamide is a co factor capable of undergoing oxidation reduction reactions and allows the transfer of thioesters Step 1 Decarboxylation of Pyruvate In the first step TPP undergoes nucleophilic attack at the C2 carbonyl of pyruvate allowing decarboxylation and the formation of the hydroxyethyl TPP intermediate This is the same mechanism employed by yeast to convert pyruvate to acetaldehyde Step 2 Acetyl Transfer from E1 to E2 In the second step the hydroxyethyl group is transferred from the E1 TPP to the E2 lipoamide forming an acetyl thioester In this process TPP is returned to the active form and the lipoamide is reduced to its dihydrolipoamide form Step 3 Acetyl Transfer to Coenzyme A In the third step the second product acetyl CoA is made following transfer of the acetyl group from dihydrolipoamide to coenzyme A The catalytic cycle is not finished as the lipoamide is reduced and must be oxidized to regenerate enzyme activity Chemical Structure of Acetyl CoA Acetyl coA is a large complicated molecule The thioester formed between the acetyl group and coenzyme A is a high energy bond which can be used as an energy source directly TCA cycle or stored for later use fatty acid biosynthesis Step 4 Oxidation of the E2 Lipoamide The E3 enzyme has two cysteines in close proximity and a permanently associated FAD molecule In step four dihydrolipoamide is oxidized by the E3 cystine disulfide bond between two Cys residues regenerating the E2 co factor and reducing the E3 disulfide in the process FAD FADH2 Co Factor Oxidation reduction of the FAD FADH2 co factor The FAD co factor can carry a single electron semiquinone form or two electrons hydroquinone form when reduced Step 5 Regeneration of the E3 Enzyme Electrons move to FAD to form FADH2 oxidizing the Cys residues and reforming the disulfide bond Electrons move from FADH2 which is tightly associated with E3 to NAD to form NADH and H E3 is restored to its oxidized state and the NADH can diffuse away oxidative phosphorylation to generate ATP Step 5 Regeneration of the E3 Enzyme The FAD Cys pair and NAD are all in close proximity in the active site of E3 which allows for the electron transfer to proceed in the desired direction Pyruvate is a Metabolic Intermediate for Multiple Pathways 1 Acetyl CoA can enter the TCA cycle or be converted into fatty acids 2 Oxaloacetate is a substrate for the TCA cycle and gluconeogenesis 3 Anaerobic pathway to regenerate NAD muscles 4 Anaerobic pathway to regenerate NAD fermentation Acetyl CoA is an Intermediate of Many Metabolic Pathways Regulation of Pyruvate Dehydrogenase Ranges from 0 all AMP to 1 all ATP Typically is maintained at 0 8 0 95 Pyruvate dehydrogenase is highly regulated by metabolites that indicate the energy charge of the cell Remember ATP changes very little but ADP and AMP concentrations can vary significantly When energy charge is high pyruvate dehydrogenase is inactivated Regulation of Pyruvate Dehydrogenase Pyruvate dehydrogenase is regulated by phosphorylation of the E1 protein When phosphorylated the E1 subunit is inactive Pyruvate dehydrogenase kinase is activated by molecules indicative of a high energy charge ATP and NADH and inactivated by molecules indicative of a low energy charge ADP and NAD The Citric Acid Cycle The citric acid cycle TCA is also commonly referred to as the tricarboxylic acid cycle and the Krebs Cycle Hans Krebs initially worked out the details of the TCA cycle and his paper detailing his findings was rejected by many major scientific journals including Nature The TCA cycle occurs in the mitochondria Metabolic Pathways are compartmentalized Glycolysis occurs in the cytoplasm TCA cycle occurs in the mitochondria Origin of Mitochondria Eukaryotes are symbiotic organisms Mitochondria are evolved from prokaryotes Mitochondria have 1 Bacterial type DNA encodes some mitochondrial proteins 2 Bacterial type ribosomes for protein synthesis 3 Double membrane similar to some bacteria Mitochondria share a common ancestor with Rickettsia prowazakeii which is the bacteria which causes the highly virulent form of Typhus Rickettsia Mitochondria TCA Cycle is an energy generating cycle Acetyl CoA is oxidized as it passes through the TCA cycle generating reduced electron carriers which can be used to synthesize ATP through oxidative phosphorylation 1 NADH 2 5 ATP 1 QH2 1 5 ATP TCA Cycle Overview of the Citric Acid Cycle In the citric acid cycle an acetyl group is condensed with oxaloacetate two CO2 are lost and oxaloacetate is regenerated Each round of the citric acid cycle generates three NADH one QH2 and one GTP