Preparation for the Citric Acid Cycle CHAPTER 18 Preparation for the Citric Acid Cycle

Overview of the Citric Acid Cycle The citric acid cycle is involved in the aerobic catabolism of carbohydrates, lipids, and amino acids Many of the intermediates of the cycle are starting points for many biosynthetic reactions: Amino acids, fatty acids, and porphyrins - Acetyl CoA is one of the key intermediates in the inter-conversion of molecules. Acetyl CoA is formed in the oxidative decarboxylation of pyruvate releasing CO2. Figures 18.1 and 18.2

Figure 18.3 Mitochondrion Glycolysis occurs within the cytosolic region of a cell. Enzymes of the citric acid cycle are in the mitochondria of eukaryotes

Transport of Pyruvate from the cytosol into the Mitochondria Pyruvate translocase transports pyruvate into the mitochondria in symport with H+ Pyruvate dehydrogenase complex

Pyruvate dehydrogenase Figure 18.4 The link between glycolysis and the citric acid cycle Pyruvate dehydrogenase complex One of the carbons in pyruvate is oxidized to CO2. The two electrons are eventually given to one NAD+

Conversion of Pyruvate to Acetyl CoA Pyruvate dehydrogenase complex is a multienzyme complex containing 3 enzymes + 5 coenzymes + other proteins Coenzymes are needed for each of these steps

Components of the PDH Complex Enzyme Coenzyme Pyruvate dehydrogenase (E1) TPP (Thiamine pyrophosphate)(Vit B1) (decarboxylase) Dihydrolipoyl Lipoic acid, HS-CoA transacetylase (E2) Dihydrolipoyl Dehydrogenase (E3) FAD, NAD+ Nicotinamide adenine dinucleotide

Conversion of Pyruvate to Acetyl CoA Decarboxylation Pyruvate dehydrogenase E1 – decarboxylase reaction involving the formation of HETPP and loss of CO2 Step 1 TPP: thymine pyrophosphate

Conversion of Pyruvate to Acetyl CoA Oxidation Pyruvate dehydrogenase E1– Oxidation reaction involving the transfer of acetyl group to the lipoamide group attached to the dihydrolipoyl transacetylase E2 Step 2 The prosthetic group, lipoic acid is attached to dihydrolipoyl transacetylase via a lysine residue. Note that the sulfur groups have been reduced. Energy rich thioester bond is formed

Conversion of Pyruvate to Acetyl CoA Formation of Acetyl CoA dihydrolipoyl transacetylase(E2): Transfer of the acetyl group to CoA forming acetyl CoA Step 3 The dihydrolipoamide prosthetic group of E2 must be reoxidized back to the lipoamide form of E2 so that further acetyl transfers can take place.

Conversion of Pyruvate to Acetyl CoA Oxidation of lipoamide Dihydrolipoyl dehydrogenase E3: oxidation back to lipoamide. The flavin adenine dinucleotide prosthetic group undergoes reduction. Steps 4 and 5 Nicotinamide adenine dinucleotide FADH2 must be re-oxidized back to FAD: this is done via reduction of NAD+

Conversion of Pyruvate to Acetyl CoA The rotating arm conversion This entire conversion can be thought of as a swinging arm of the lipoamide prosthetic group of E2. Figure 13.1 Reactions of the pyruvate dehydrogenase complex. The lipoamide prosthetic group (blue) is attached by an amide linkage between lipoic acid and the side chain of a lysine residue of This prosthetic group is a swinging arm that carries the two-carbon unit from the pyruvate dehydrogenase active site to the dihydrolipoamide acetyltransferase active site. The arm then carries hydrogen to the dihydrolipoamide dehydrogenase active site. Ensures the product of one step does not diffuse into the medium Figure 18.7 Reactions of the PDH complex

Regulation of the pyruvate dehydrogenase complex

(The Kinase and Phosphatase are regulated) Figure 18.9 Regulation of mammalian PDH complex occurs predominately by covalent modification (The Kinase and Phosphatase are regulated) High ATP, acetyl CoA, NADH High ADP, pyruvate Figure 13.12 Regulation of the mammalian pyruvate dehydrogenase complex by phosphorylation of the E1 component. The regulatory kinase and phosphatase are both components of the mammalian complex. The kinase is activated by NADH and acetyl CoA, products of the reaction catalyzed by the pyruvate dehydrogenase complex, and inhibited by ADP and the substrates pyruvate, NAD+, and HS–CoA. Dephosphorylation is stimulated by elevated levels of Ca2+. Metal activated enzyme

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