Pyruvate generated in the cytosol by glycolysis represents a node in the metabolism of carbohydrates, proteins, and fats. Under anaerobic conditions, pyruvate may simply be reduced to lactate in the cytosol, regenerating for continued ATP production by glycolysis. Pyruvate may serve as a precursor for the synthesis of amino acids (Chapter 22). In eukaryotes, pyruvate may diffuse into mitochondria, first through large openings in the outer mitochondrial membrane and then into the matrix via an -coupled pyruvate-specific symporter in the inner mitochondrial membrane, the mitochondrial pyruvate carrier (MPC). Pyruvate that enters mitochondria may be oxidized by the citric acid cycle to generate energy or, after conversion to acetyl-CoA, may be used as the starting material for synthesis of fatty acids and sterols.

Pyruvate in the mitochondrial matrix is oxidized to acetyl-CoA and by the pyruvate dehydrogenase (PDH) complex, a highly ordered cluster of enzymes and cofactors. In the PDH complex, a series of chemical intermediates remain bound to the enzyme subunits as a substrate (pyruvate) is transformed into the final product (acetyl-CoA). Five cofactors, four of which are derived from vitamins, participate in the reaction mechanism. The regulation of this enzyme complex illustrates how a combination of covalent modification and allosteric mechanism results in precisely regulated flux through a metabolic step.

The overall reaction catalyzed by the pyruvate dehydrogenase complex is an oxidative decarboxylation, an irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of and the two remaining carbons become the acetyl group of acetyl-CoA (Fig. 16-3). The NADH formed in this reaction gives up a hydride ion to the respiratory chain (Fig. 16-1), which carries the two electrons to oxygen or, in anaerobic microorganisms, to an alternative electron acceptor such as nitrate or sulfate. The transfer of electrons from NADH to oxygen ultimately generates 2.5 molecules of ATP per pair of electrons. The irreversibility of the PDH complex reaction has been demonstrated by isotopic labeling experiments: the complex cannot reattach radioactively labeled to acetyl-CoA to yield carboxyl-labeled pyruvate.

The combined dehydrogenation and decarboxylation of pyruvate to the acetyl group of acetyl-CoA (Fig. 16-3) requires the sequential action of three different enzymes and five different coenzymes or prosthetic groups — thiamine pyrophosphate (TPP), lipoate, coenzyme A (CoA, sometimes denoted CoA-SH, to emphasize the role of the group), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NAD). We encountered TPP as the coenzyme of pyruvate decarboxylase (see Fig. 14-13). Lipoate (Fig. 16-4), has two thiol groups that can undergo reversible oxidation to a disulfide bond , similar to that between two Cys residues in a protein. Because of its capacity to undergo oxidation-reduction reactions, lipoate can serve both as an electron (hydrogen) carrier and as an acyl carrier, as we shall see. CoA serves as the carrier of the activated acyl group. We described the roles of FAD and NAD as electron carriers in Chapter 13.

The PDH complex contains multiple copies of three enzymes — pyruvate dehydrogenase , dihydrolipoyl transacetylase , and dihydrolipoyl dehydrogenase that catalyze the oxidation of pyruvate. The number of copies of each enzyme and therefore the size of the complex varies among species. A central core is formed of many copies (24 to 60) of surrounded by multiple and variable numbers of copies of and (Fig. 16-5). Two regulatory proteins are also part of the complex: a protein kinase and a phosphoprotein phosphatase, discussed below.

The active site of has noncovalently bound TPP. has the prosthetic group lipoate, attached through an amide bond to the ε-amino group of a Lys residue (Fig. 16-4). has three functionally distinct domains: one or more (depending on the species) amino-terminal lipoyl domains, containing the lipoyl-Lys residue(s); the central - and -binding domain; and the inner-core acyltransferase domain, which contains the acyltransferase active site. The domains of are separated by linkers, sequences of 20 to 30 amino acid residues, rich in Ala and Pro and interspersed with charged residues; these linkers tend to assume their extended forms, holding the three domains apart. The attachment of lipoate to the end of a Lys side chain in produces a long, flexible arm that can move from the active site of to the active sites of and , a distance of perhaps 5 nm or more, while holding the intermediate captive throughout the reaction sequence. has tightly bound FAD.

This basic structure of the PDH complex is seen in two other enzyme complexes that catalyze similar reactions: α-ketoglutarate dehydrogenase, which oxidizes α-ketoglutarate in the citric acid cycle (described below), and the branched-chain α-keto acid dehydrogenase, which oxidizes α-keto acids derived from the breakdown of the branched-chain amino acids valine, isoleucine, and leucine (see Fig. 18-28). Within a given species, is identical in all three complexes. The remarkable similarity in protein structure, cofactor requirements, and reaction mechanisms of these three complexes doubtless reflects a common evolutionary origin; they are paralogs.

Figure 16-6 shows schematically how the pyruvate dehydrogenase complex carries out the five consecutive reactions in the decarboxylation and dehydrogenation of pyruvate without allowing the intermediates to leave the surface of the complex. Step is essentially identical to the reaction catalyzed by pyruvate decarboxylase (see Fig. 14-13c); C-1 of pyruvate is released as , and C-2, which in pyruvate has the oxidation state of an aldehyde, is attached to TPP as a hydroxyethyl group. This first step is the slowest and therefore limits the rate of the overall reaction. In step the hydroxyethyl group is oxidized to the level of a carboxylic acid (acetate). The two electrons removed in this reaction reduce the of a lipoyl group on to two thiol groups. The acetyl moiety produced in this oxidation-reduction reaction is first esterified to one of the lipoyl groups, then transesterified to CoA to form acetyl-CoA (step ). Thus the energy of oxidation drives the formation of a high-energy thioester of acetate. The remaining reactions catalyzed by the PDH complex (by , in steps and ) are electron transfers necessary to regenerate the oxidized (disulfide) form of the lipoyl group of , to prepare the enzyme complex for another round of oxidation. The electrons removed from the hydroxyethyl group derived from pyruvate pass through FAD to , forming NADH, which can enter the respiratory chain.

The five-reaction sequence shown in Figure 16-6 is an example of substrate channeling. The swinging lipoyllysyl arms of accept from the two electrons and the acetyl group that it derived from pyruvate, and pass them to . The intermediates of the multistep sequence never leave the complex, and the local concentration of the substrate of is kept very high. This substrate channeling also prevents theft of the activated acetyl group by other enzymes that use this group as substrate. We will encounter other enzymes that use a similar tethering mechanism to channel substrate between active sites, with lipoate, biotin, or a CoA-like moiety serving as cofactor.

Four different vitamins required in human nutrition are vital components of the pyruvate dehydrogenase complex: thiamine (TPP), pantothenate (CoA), riboflavin (FAD), and niacin (NAD). As one might predict, mutations in the genes for the subunits of the PDH complex, or a dietary vitamin deficiency, can have severe consequences. Thiamine-deficient animals are unable to oxidize pyruvate normally. This is of particular importance to the brain, which usually obtains all its energy from the aerobic oxidation of glucose in a pathway that necessarily includes the oxidation of pyruvate. Beriberi, a disease that results from thiamine deficiency, is characterized by loss of neural function. This disease occurs primarily in populations that rely on a diet consisting mainly of white (polished) rice, which lacks the hulls that contain most of the thiamine found in rice. People who habitually consume large amounts of alcohol can also develop thiamine deficiency, because much of their dietary intake consists of the vitamin-free “empty calories” of distilled spirits. An elevated level of pyruvate in the blood is often an indicator of defects in pyruvate oxidation due to one of these causes.