Question

22. The pyruvate dehydrogenase complex provides a transition between glycolysis and the citric
acid cycle. Which co-factors are considered stoichiometric and which are considered
catalytic?
23. Which conditions exert a negative influence (reduce the activity) of pyruvate dehydrogenase?
24. What is the critical role for lipoic acid in the pyruvate dehydrogenase complex?
25. Central to the function of the citric acid cycle are two decarboxylation reactions. What are
these decarboxylation reaction?
26. Your friend tells you of a celebrity touting a new “miracle diet drug” which targets succinyl
CoA synthetase. The drug acts by stabilizing or locking in the succinyl-phosphate
intermediate state. Why would this drug cause serious side effects?

Answer 1

22. There are five co-factors for the PDH complex, thiamine pyrophosphate (TPP), lipoic acid, FAD, CoA, and NAD+. TPP, lipoic acid, and FAD are catalytic co-factors while CoA and NAD+ are stoichiometric co-factors and consumed by the PDH reactions. Catalytic co-factors are not consumed by the enzyme-catalyzed reaction. Their concentrations are the same at the end of the reaction. In contrast, stoichiometric co-factors are consumed as the reaction proceeds and they appear in the products of the reaction.

23. Pyruvate dehydrogenase is inhibited when one or more of the three following ratios are increased: ATP/ADP, NADH/NAD+ and acetyl-CoA/CoA.

In eukaryotes PDC is tightly regulated by its own specific pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP), deactivating and activating it respectively.

PDK phosphorylates three specific serine residues on E1 with different affinities. Phosphorylation of any one of them renders E1 (and in consequence the entire complex) inactive.

Dephosphorylation of E1 by PDP reinstates complex activity.

Products of the reaction act as allosteric inhibitors of the PDC, because they activate PDK. Substrates in turn inhibit PDK, and thus, reactivating PDC.

During starvation, PDK increases in amount in most tissues, including skeletal muscle, via increased gene transcription. Under the same conditions, the amount of PDP decreases. The resulting inhibition of PDC prevents muscle and other tissues from catabolizing glucose and gluconeogenesis precursors. Metabolism shifts toward fat utilization, while muscle protein breakdown to supply gluconeogenesis precursors is minimized, and available glucose is spared for use by the brain.

24. At least two additional enzymes regulate the activity of the complex.

The conversion of pyruvate into acetyl CoA consists of three steps: decarboxylation, oxidation, and transfer of the resultant acetyl group to CoA.

These steps must be coupled to preserve the free energy derived from the decarboxylation step to drive the formation of NADH and acetyl CoA. First, pyruvate combines with TPP and is then decarboxylated. This reaction is catalyzed by the pyruvate dehydrogenase component (E1) of the multienzyme complex. A key feature of TPP, the prosthetic group of the pyruvate dehydrogenase component, is that the carbon atom between the nitrogen and sulfur atoms in the thiazole ring is much more acidic than most =CH- groups, with a pKa value near 10. This center ionizes to form a carbanion, which readily adds to the carbonyl group of pyruvate.

This addition is followed by the decarboxylation of pyruvate. The positively charged ring of TPP acts as an electron sink that stabilizes the negative charge that is transferred to the ring as part of the decarboxylation. Protonation yields hydroxyethyl-TPP.

Second, the hydroxyethyl group attached to TPP is oxidized to form an acetyl group and concomitantly transferred to lipoamide, a derivative of lipoic acid that is linked to the side chain of a lysine residue by an amide linkage.

The oxidant in this reaction is the disulfide group of lipoamide, which is reduced to its disulfhydryl form. This reaction, also catalyzed by the pyruvate dehydrogenase component E1, yields acetyllipoamide.

Third, the acetyl group is transferred from acetyllipoamide to CoA to form acetyl CoA.

Dihydrolipoyl transacetylase (E2) catalyzes this reaction. The energy-rich thioester bond is preserved as the acetyl group is transferred to CoA. Recall that CoA serves as a carrier of many activated acyl groups, of which acetyl is the simplest . Acetyl CoA, the fuel for the citric acid cycle, has now been generated from pyruvate.

The pyruvate dehydrogenase complex cannot complete another catalytic cycle until the dihydrolipoamide is oxidized to lipoamide. In a fourth step, the oxidized form of lipoamide is regenerated by dihydrolipoyl dehydrogenase (E3). Two electrons are transferred to an FAD prosthetic group of the enzyme and then to NAD+.

This electron transfer to FAD is unusual, because the common role for FAD is to receive electrons from NADH. The electron transfer potential of FAD is altered by its association with the enzyme and enables it to transfer electrons to NAD+. Proteins tightly associated with FAD or flavin mononucleotide (FMN) are called flavoproteins.

25.

1) the oxidative decarboxylation of isocitrate to ?-ketoglutarate (isocitrate dehydrogenase),

2) the oxidative decarboxylation of ?-ketoglutarate to succinyl CoA (?-ketoglutarate dehydrogenase complex),

26. SCS is a Krebs Cycle enzyme that catalyzes substrate-level phosphorylation in the forward direction (5) and replenishes succinyl-CoA for ketone body catabolism and porphyrin biosynthesis in the reverse direction.

A phosphate group is substituted for coenzyme A, and a high- energy bond is formed. This energy is used in substrate-level phosphorylation (during the conversion of the succinyl group to succinate) to form either guanine triphosphate (GTP) or ATP. There are two forms of the enzyme, called isoenzymes, for this step, depending upon the type of animal tissue in which they are found. One form is found in tissues that use large amounts of ATP, such as heart and skeletal muscle. This form produces ATP. The second form of the enzyme is found in tissues that have a high number of anabolic pathways, such as liver. This form produces GTP. GTP is energetically equivalent to ATP; however, its use is more restricted. In particular, protein synthesis primarily uses GTP.