Dehydrogenate complex

A dehydrogenating complexation of the 2- and 3-borolenes C4H5BR (R = Me, Ph) also occurs with [Rh(7j -C2H4)2C1]2, forming a triple-decked 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. 

Searls and Sanadi (1960a) determined the reduction potential of the pig heart dihydrolipoic dehydrogenase from the extent of its reduction at different DPNH DPN ratios. The value is between —0.332 and —0.320 volt at pH 7.0 and 25°C. Thus the reduction potential of the flavoprotein is close to that of the DPNH-DPN system, —0.320 volt at pH 7.0 and 25°C (Burton and Wilson, 1953), and the Lip(SH)2-LipS2 system, —0.325 volt at pH 7.0 and 25°C (see Section II, A). This is consistent with the ready reversibility of reaction (26) as well as the high initial reaction rates in both directions. It would appear that DPN is the physiological electron acceptor for the a-keto acid dehydrogenation complexes in vivo and that the DPNH formed is reoxidized by way of the electron transport chain. However, the... 

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-... 

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