Pyruvate dehydrogenation complex

The decarboxylation reaction, Eq. (7), is visualized as a cleavage of the a-keto acid to yield CO2 and an enzyme-bound aldehyde-thiamine pyrophosphate (RCHO—TPP) compound, i.e., active aldehyde. There is now unequivocal evidence for this reaction since a pyruvic carboxylase (El) has been shown to be an essential component of the E. coli pyruvate dehydrogenation complex (Koike and Reed, 1961 Gounaris and Hager, 1961) and the nature of the aldehyde-TPP compound has been elucidated (Breslow, 1958 Breslow and McNelis, 1959 Krampitz et al., 1961 Holzer and Beaucamp, 1961 Carlson and Brown, 1961). 

The acyl-generation reaction, Eq. (8), has been visualized as a reductive acylation of protein-bound lipoic acid. As will be seen below, this reaction is now belitwod to consist of two steps an oxidation of the 2-hydroxyalkyl-thiamine pyrophcjsphatc to 2-aoylthiaminc pyrophosphate with a concomitant reduction of bound lipoic acid, and a transfer of (he acyl group of 2-acylthiamine pyrophosphate to the bound dihydrolipoic acid (Das el al., 19(il). An enzymatic component which contains bound lipoic acid and apparently catalyzes reactions (8) and (9) has been isolated from the E. mli pyruvate dehydrogenation complex (Koike and Reed, 1961). This component, designated lipoyl-Ea in Fig. 1, has been tentatively named lipoic reductase-transacetylase. 

Support for the proposal that lipoic acid is bound in the pyruvate dehydrogenation complex in covalent linkage through its carboxyl group was furnished by studies with a hydrolytic enzyme, lipoyl-X hydrolase, obtained from extracts of S. faecalis (Reed et al., 1958b). Incubation of the E. coli pyruvate and a-ketoglutarate dehydrogenation complexes with lipoyl-X hydrolase released approximately 96% of the bound lipoic acid (Koike and Reed, 1960) and resulted in a loss of the DPN-linked a-keto... 

The amino acid sequence about the iM -lipoyUysine residue in the two enzyme complexes has been determined (Daigo and Reed, 1962b). The sequence Gly-Asp e-Lipoyl-Lys AIa is present in the pyruvate dehydrogenation complex and the sequence Thr-Asp-e-Lipoyl-Lys-Val-(Val,Leu)-Glu is present in the a-ketoglutarate dehydrogenation complex. It is thus apparent that both complexes contain the sequence Asp - -Lipoyl-Lys, but otherwise the sequences arc different. This difference in amino acid sequence is probably responsible, at least in part, for the substrate specificity of the two complexes. 

The resolution and reconstitution experiments described above indicate that the E. coli pyruvate dehydrogenation complex is a highly organized multienzyme unit. The number of molecules of carboxylase and of flavoprotein per molecule of complex have been calculated. Those numbers are about 12 for the carboxylase and about 6 for the flavoprotein. Each mole-... 

The results of the resolution and reconstitution experiments carried out with the E. coli pyruvate dehydrogenation complex indicate that there are specific binding sites on the lipoic reductase-transacetylase component for the carboxylase and the flavoprotein. In other words, the latter two enzymes appear to be specifically oriented with respect to the lipoic acid bound to the lipoic reductase-transacetylase component. It is evident from the re-... 

Fi(i. 8. A schematic representation of po.ssible interactions between several lipoyllysyl moieties in the E. coli pyruvate dehydrogenation complex. These interactions may involve thiol-disulfide interchange and acetyl transfer. The arrows describe the area covered by each lipoyllysyl moiety. 

Figure 16-6 shows schematically how the pyruvate dehydrogenase complex carries out the five consecutive reactions in the decarboxylation and dehydrogenation of pyruvate. Step CD is essentially identical to the reaction catalyzed by pyruvate decarboxylase (see Fig. 14-13c) C-l of pyruvate is released as C02, 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 car-...

Fkj. 6. Functional form of lipoic acid in Escherichia coli pyruvate and -koto-glutarate dehydrogenation complexes. The earboxyl group of lipoie acid is bound in amide linkage to the e-amino group of a lysine residue, providing a flexible arm of approximately 14 A for the reactive dithiolane ring. 

Fig. 60. The respiratory chain of higher plants. Ubiquinone appears to serve as an electron reservoir. = probable site of ATP formation. SD = succinate dehydrogenase. It used to be assumed that, with the exception of the reaction catalyzed by SD, the hydrogen acceptor in dehydrogenation reactions was NAD+ and that the hydrogen then entered the respiratory chain in the form of NADH+H+. In reality the situation is more complicated since the lipoic acid oxidizing flavoproteid of the pyruvate dehydrogenase and the a-ketoglutarate dehydrogenase complexes—in both cases the same flavoproteid is involved—can establish direct contact with the flavoproteins of the respiratory chain just like succinate dehydrogenase. associated with encircled flavoproteins means that ATP can be formed as a result of transitions between the various flavoproteins, except those involving SD.

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