Dihydrolipoyl dehydrogenase reaction catalyzed

The reaction of hydroxyethyl-TPP with the oxidized form of lipoic acid yields the energy-rich thiol ester of reduced lipoic acid and results in oxidation of the hydroxyl-carbon of the two-carbon substrate unit (c). This is followed by nucleophilic attack by coenzyme A on the carbonyl-carbon (a characteristic feature of CoA chemistry). The result is transfer of the acetyl group from lipoic acid to CoA. The subsequent oxidation of lipoic acid is catalyzed by the FAD-dependent dihydrolipoyl dehydrogenase and NAD is reduced. 

The pyruvate dehydrogenase complex from Escherichia coli is considerably more complex than tryptophan synthetase. It has a molecular weight of approximately 4.6 millon and contains three enzymes pyruvate dehydrogenase (Et), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3).82 The overall reaction catalyzed by the complex is... 

Further transfer of the acyl group to coenzyme A is catalyzed by the same enzyme. This displacement reaction produces reduced lipoic acid. A third enzyme, dihydrolipoyl dehydrogenase, catalyzes oxidation of this product back to the disulfide form. The electrons lost in that oxidation are transferred first to an enzyme-bound flavin (not shown in the figure) and then to NAD +. ... 

The conversion of pyruvate to acetyl-CoA. The reactions are catalyzed by the enzymes of the pyruvate dehydrogenase complex. This complex has three enzymes pyruvate decarboxylase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. In addition, five coenzymes are required thiamine pyrophosphate, lipoic acid, CoASH, FAD, and NAD+. Lipoic acid is covalently attached to... 

In the reaction catalyzed by dihydrolipoyl dehydrogenase, one of three enzymes in the pyruvate dehydrogenase complex (fig. 13.5), electrons flow from oxidized lipoic acid to enzyme-bound FAD to NAD+. Compare the flow of electrons in the latter part of this scheme (FAD to NAD + ) to the flow of electrons in the electron transport scheme (Complex I). Is there a distinct difference in the flow of electrons in the two schemes If so, can you provide a possible explanation for this difference ... 

Note that in the reactions of dihydrolipoyl transacetylase the lipoamide has been reduced from a disulphide to two sulphhydryl groups. In order to continue operation lipoamide must be reoxidized and that is accomplished by the final enzyme of the complex, dihydrolipoyl dehydrogenase. The reactions catalyzed by this enzyme are complex, but the net result is the transfer of two electrons from the lipoamide to NAD+ to give NADH. 

A FIGURE 3-21 Structure and function of pyruvate dehydrogenase, a large multimeric enzyme complex that converts pyruvate into acetyl CoA. (a) The complex consists of 24 copies of pyruvate decarboxylase (Ei), 24 copies of lipoamide transacetylase (E2), and 12 copies of dihydrolipoyl dehydrogenase (E3). The El and E3 subunits are bound to the outside of the core formed by the E2 subunits, (b) The reactions catalyzed by the complex include several enzyme-bound intermediates (not shown). The tight structural integration of the three enzymes increases the rate of the overall reaction and minimizes possible side reactions. 

Pyruvate dehydrogenase complex. Allen et al.(1964) and later de Vries et al.(1973) postulated that this complex in P. shermanii includes pyruvate dehydrogenase that uses thiamine diphosphate as coenzyme, dihydrolipoyl transacetylase containing lipoic acid, and dihydrolipoyl dehydrogenase containing NAD and FAD. The complex catalyzes the following reaction ... 

MECHANISM PROPOSED FOR THE REACTION CATALYZED BY DIHYDROLIPOYL DEHYDROGENASE... 

This enzyme complex [EC 1.2.4.4], also known as 3-methyl-2-oxobutanoate dehydrogenase (lipoamide) and 2-oxoisovalerate dehydrogenase, catalyzes the reaction of 3-methyl-2-oxobutanoate with lipoamide to produce S-(2-methylpropanoyl)dihydrolipoamide and carbon dioxide. Thiamin pyrophosphate is a required cofactor. The complex also can utilize (5)-3-methyl-2-oxopenta-noate and 4-methyl-2-oxopentanoate as substrates. The complex contains branched-cham a-keto acid decarboxylase, dihydrolipoyl acyltransferase, and dihydrolipoa-mide dehydrogenase [EC 1.8.1.4]. 

Figure 17.9. Reactions of the Pyruvate Dehydrogenase Complex. At the top (center), the enzyme (represented by a yellow, a blue, and two red spheres) is unmodified and ready for a catalytic cycle. (1) Pyruvate is decarboxylated to form the hydroxy ethyl TPP. (2) The dihydrolipoyl arm of E2 moves into the active site of E. (3) Ej catalyzes the transfer of the two-carbon group to the dihydrolipoyl group to form the acetyl-lipoyl complex. (4) E2 catalyzes the transfer of the acetyl moiety to CoA to form the product acetyl CoA. The disulfhydryl lipoyl arm then swings to the active site of E3. E3...

The behavior of lipoamide dehydrogenase in the intact pyruvate dehydrogenase complex has been investigated. The multienzyme complex consists of 24 pyruvate decarboxylase subunits, 24 dihydrolipoyl transacetylase subunits, and 12 lipoamide dehydrogenase subunits. The complex catalyzes a series of reactions beginning with pyruvate decarboxylation, followed by transfer of an acetyl moiety to CoA, and finally the oxidation of dihydrolipoamide by NAD. Stopped-flow smdies showed that lipoamide dehydrogenase in the complex behaves largely the same as it does when studied alone. 

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