Table 1. Pyruvate dehydrogenase complex of E. coli

Pyruvate dehydrogenase is a member of a family of homologous complexes that includes the citric acid cycle enzyme α-ketoglutarate dehydrogenase, a branched-chain α-ketoacid dehydroge?nase, and acetoin dehydrogenase, found in certain prokaryotes. These complexes are giant, with molecular masses ranging from 4 to 10 million daltons. As we will see, their elaborate structures allow groups to travel from one active site to another, connected by tethers to the core of the structure. The mechanism of the pyruvate dehydrogenase reaction is wonderfully complex, more so than is suggested by its relatively simple stoichiometry. The reaction requires the participation of the three enzymes of the pyruvate dehydrogenase complex, each composed of several polypeptide chains, and five coenzymes: thiamine pyrophosphate (TPP), lipoic acid, and FAD serve as catalytic cofactors, and CoA and NAD⁺ are stoichiometric cofactors.

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 at?tached 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.

Although the structure of an intact member of the pyruvate dehydrogenase complex family has not yet been determined in atomic detail, the structures of all of the component enzymes are now known, albeit from different complexes and species. Thus, it is now possible to construct an atomic model of the complex to understand its activity.

The core of the complex is formed by E2. Acetyltransferase consists of eight catalytic trimers assembled to form a hollow cube. Each of the three subunits forming a trimer has three major domains. At the amino terminus is a small domain that contains a bound lipoamide cofactor attached to a lysine residue. This domain is homologous to biotin-binding domains such as that of pyruvate carboxylase. The lipoamide domain is followed by a small domain that interacts with E3 within the complex. A larger transacetylase domain completes an E2 subunit. E1 is an α2β2 tetramer, and E3 is a αβ dimer. Twenty-four copies of E1 and 12 copies of E3 surround the E2 core. How do the three distinct active sites work in concert?

1. Pyruvate is decarboxylated at the active site of E1, forming the substituted TPP intermediate, and CO₂ leaves as the first product. This active site lies within the E1 complex, connected to the enzyme surface by a 20-Å-long hydrophobic channel.

2. E2 inserts the lipoyl-lysine arm of the lipoamide domain into the channel in E1.

3. E1 catalyzes the transfer of the acetyl group to the lipoamide. The acetylated lipoyl-lysine arm then leaves E1 and enters the E2 cube through 30 Å windows on the sides of the cube to visit the active site of E2, located deep in the cube at the subunit interface.

4. The acetyl moiety is then transferred to CoA, and the second product, acetyl CoA, leaves the cube. The reduced lipoyl-lysine arm then swings to the active site of the E3 flavoprotein.

5. At the E3 active site, the lipoamide acid is oxidized by coenzyme FAD.

6. The final product, NADH, is produced with the reoxidation of FADH₂, and the reactivated lipoamide is ready to begin another reaction cycle.

The structural integration of three kinds of enzymes makes the coordinated catalysis of a complex reaction possible. The proximity of one enzyme to another increases the overall reaction rate and minimizes side reactions. All the intermediates in the oxidative decarboxylation of pyruvate are tightly bound to the complex and are readily transferred because of the ability of the lipoyl-lysine arm of E2 to call on each active site in turn.

Date: 2016-06-13; view: 227