Production of Acetyl-CoA (Activated Acetate):- In Substrate Channeling, Intermediates Never Leave the Enzyme Surface
Figure 16–6 shows schematically how the pyruvate de hydrogenase complex carries out the five consecutive reactions in the decarboxylation and dehydrogenation of pyruvate. Step 1 is essentially identical to the reaction catalyzed by pyruvate decarboxylase (see Fig. 14–13c); C-1 of pyruvate is released as CO2, 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. It is also the point at which the PDH complex exercises its substrate specificity. In step 2 the hydroxyethyl group is oxidized to the level of a carboxylic acid (acetate). The two electrons removed in this reaction reduce the OSOSO of a lipoyl group on E2 to two thiol (-SH) groups. The acetyl moiety produced in this oxidation-reduction reaction is first esterified to one of the lipoyl OSH groups, then trans esterified to CoA to form acetyl-CoA (step 3). Thus, the energy of oxidation drives the formation of a high energy thioester of acetate. The remaining reactions catalyzed by the PDH complex (by E3, in steps 4 and 5) are electron transfers necessary to regenerate the oxidized (disulfide) form of the lipoyl group of E2 to prepare the enzyme complex for another round of oxidation. The electrons removed from the hydroxyethyl group derived from pyruvate pass through FAD to NAD+.
Central to the mechanism of the PDH complex are the swinging lipoyllysyl arms of E2, which accept from E1 the two electrons and the acetyl group derived from pyruvate, passing them to E3. All these enzymes and coenzymes are clustered, allowing the intermediates to react quickly without diffusing away from the sur face of the enzyme complex. The five-reaction sequence shown in Figure 16–6 is thus an example of substrate channeling. The intermediates of the multistep sequence never leave the complex, and the local concentration of the substrate of E2 is kept very high. Channeling also prevents theft of the activated acetyl group by other enzymes that use this group as substrate. As we shall see, a similar tethering mechanism for the channeling of substrate between active sites is used in some other enzymes, with lipoate, biotin, or a CoA-like moiety serving as cofactors. As one might predict, mutations in the genes for the subunits of the PDH complex, or a dietary thiamine 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 in which most of the thiamine of rice is found. 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.
FIGURE 16–6 Oxidative decarboxylation of pyruvate to acetyl-CoA by the PDH complex. The fate of pyruvate is traced in red. In step 1 pyruvate reacts with the bound thiamine pyrophosphate (TPP) of pyruvate dehydrogenase (E1), undergoing decarboxylation to the hydroxyethyl derivative (see Fig. 14–13). Pyruvate dehydrogenase also carries out step 2, the transfer of two electrons and the acetyl group from TPP to the oxidized form of the lipoyllysyl group of the core en zyme, dihydrolipoyl transacetylase (E2), to form the acetyl thioester of the reduced lipoyl group. Step 3 is a transesterification in which the OSH group of CoA replaces the OSH group of E2 to yield acetyl-CoA and the fully reduced (dithiol) form of the lipoyl group. In step 4 dihydrolipoyl dehydrogenase (E3) promotes transfer of two hydrogen atoms from the reduced lipoyl groups of E2 to the FAD prosthetic group of E3, restoring the oxidized form of the lipoyllysyl group of E2. In step 5 the reduced FADH2 of E3 transfers a hydride ion to NAD+, forming NADH. The enzyme complex is now ready for another catalytic cycle. (Subunit colors correspond to those in Fig. 16–5b.)
