Lipoic acid transacetylase

Figure 17-5. Oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex. Lipoic acid is joined by an amide link to a lysine residue of the transacetylase component of the enzyme complex. It forms a long flexible arm, allowing the lipoic acid prosthetic group to rotate sequentially between the active sites of each of the enzymes of the complex. (NAD nicotinamide adenine dinucleotide FAD, flavin adenine dinucleotide TDP, thiamin diphosphate.)...

An acyl-transfer and redox coenzyme containing two sulfhydryl groups that form a dithiolane ring in the oxidized (disulfide) form. The redox potential at pH 7 is -0.29 volts. Lipoic acid is attached to the e-amino group of lysyl residues of transacetylases (subunit of a-ketoacid dehydrogenase complexes), thereby permitting acyl... 

Dihydrolipoyl transacetylase transfers the acetyl CoA to its lipoic acid... 

Figure 7-1. Conversion of pyruvate to acetyl CoA by the pyruvate dehydrogenase complex. The three enzymes, pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase, exist in a complex associated with the mitochondrial matrix. Each enzyme requires at least one coenzyme that participates in the reaction. TPP, thiamine pyrophosphate Lip, lipoic acid CoA, coenzyme A.

FIGURE 16-4 Lipoic acid (lipoate) in amide linkage with a Lys residue. The lipoyllysyl moiety is the prosthetic group of dihydrolipoyl transacetylase (E2 of the PDH complex). The lipoyl group occurs in oxidized (disulfide) and reduced (dithiol) forms and acts as a carrier of both hydrogen and an acetyl (or other acyl) group. 

The hydroxyethyl intermediate is oxidized by transfer to the disulfide form of lipoic acid covalently bound to dihydrolipoyl transacetylase. —... 

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... 

The lipoic acid is covalently attached to the transacetylase (E2). It acts as a swinging arm , interacting with pyruvate decarboxylase (E ) to accept the hydroxyethyl derivative of pyruvate, with CoA to produce acetyl-CoA, and with dihydrolipoyl dehydrogenase (E3) so that it can be reoxidized. 

Lipoamide Lipoic acid is linked through an amide bond to the side-chain NH2 group of a lysine residue in dihydrolipoyl transacetylase... 

Dihydrolipoyl transacetylase (E2) Catalyzes transfer of acetyl group to CoASH 24 (60) Lipoic acid, CoASH... 

The third step of the reaction is also catalyzed by dihydrolipoyl transacety-lase. A molecule of GoA-SH attacks the thioester linkage, and the acetyl group is transferred to it. The acetyl group remains bound in a thioester linkage this time it appears as acetyl-GoA rather than esterified to lipoic acid. The reduced form of lipoic acid remains covalently bound to dihydrolipoyl transacetylase (see Figure 19.4). The reaction of pyruvate and GoA-SH has now reached the stage of the products, carbon dioxide and acetyl-GoA, but the lipoic acid coenzyme is in a reduced form. The rest of the steps regenerate the lipoic acid, so further reactions can be catalyzed by the transacetylase. 

Five enzymes are involved in the pyruvate dehydrogenase complex of mammals. Pyruvate dehydrogenase transfers a two-carbon unit to TPP and releases CO. Dihydrolipoyl transacetylase transfers the two-carbon acetyl unit to lipoic acid and then to coenzyme A. Dihydrolipoyl dehydrogenase reoxidizes lipoic acid and reduces NAD" to NADH. Pyruvate dehydrogenase kinase phosphorylates PDH. PDH phosphatase removes the phosphate. Lipoic acid plays a role both in redox and in acetyl-transfer reactions. 

Transfer of the 2-carbon unit to Coenzyme A. This reaction is carried out by the dihydrolipamide transacetylase (E2) component of the complex. Lipoic acid is an 8-carbon carboxylic acid with a disulfide bond linking the 6 and 8 carbons ... 

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. 

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-... 

The really unique reaction of the lipoate centre in ct-keto acid metabolism is the oxidative thioester formation from a thiamine-coordinated active aldehyde . Thiol transacetylase and dithiol-disulphide oxidation reduction roles are well-known attributes of other biological thiols. Unfortunately mechanistic studies on this reductive acylation of a cyclic disulphide have so far received little attention. Proposals that a lipoic acid-thiamine pyrophosphate compound was the functional entity in a-keto acid oxidation have been completely abandoned, but data supporting this concept remain unexplained. Investigations in this area might have some relevance for the reductive acylation process. 

Pyruvate dehydrogenase complex is a multienzyme complex which converts pyruvate to acetyl CoA. It consists of three enzymes pyruvate dehydrogenase, dihydrolipoyl transacetylase. And dihydrolipoyl dehydrogenase. It has five coenzymes NAD, CoA, lipolc acid, thiamine P3Tophosphate. and FAD. The enzyme dihydrolipoyl dehydrogenase is centrally placed m the crystal. It possesses a lipoic acid moiety attached to a lysine and it is hypothesized that u is this structure that moves around the crystal of tae enzyme as a swinging arm bringing successive intermediates to the other enzymes in order that the reaction proceeds Do we have any experimental support for this hypothesis ... 

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 ... 

The thiol transacetylase that catalyzes this reaction has been partially purified from extracts of E. coli by Hager and Gunsalus (personal communication), and was shown to be relatively specific. We have recently discovered enzymes in extracts of C. kluyveri (47) and in pigeon-liver extracts (48) that catalyze similar acetyl transfers from acetyl-SCoA to 2-mercap-toethanol, thioglycollate, reduced lipoic acid, and hydrogen sulfide however, GSH and cysteine are relatively inert as acetyl acceptors. It remains to be demonstrated if one or more enzymes are involved in these acyl-transfer reactions. The enzyme-catalyzed reactions are to be differentiated from the nonenzymatic reactions in that they occur readily in more dilute solutions and also at neutral to slightly acid pH. 

Reaction 3 shows the transfer of an acyl (acetyl) group to CoA. In the presence of Pi and phosphotransacetylase, the acyl group would accumulate as acetyl phosphate. The transacetylase reaction of lipoic acid has been determined by employing the reverse of the phosphotransacetylase reaction, mentioned above, and reaction 3 of Fig. 4. In the presence of catalytic amounts of CoA, lipoic transacetylase activity can be demonstrated by measuring the accumulation of a heat-stable hydroxamic acid derived from the acyl-Iipoate or by measuring the disappearance of —SH groups. 

Oxoglutarate undergoes oxidative decarboxylation to succinyl-CoA, via multi-enzyme reaction similar to the reaction pattern of pyruvate. The multi-enzyme complex (mw about 2 x 10 ) is an octamer of an elementary unit containing each of the three contributing enzyme proteins oxoglutarate decarboxylase, dihydro-lipoyl transacetylase, and dihydrolipoyl dehydrogenase. The overall reaction involves thiamine pyrophosphate, lipoic acid, CoASH and NAD succinyl-CoA is the end product ... 

The aim of the present experiment was to study the PDHc activity in the livers and brains of developing rats born to females kept on a fat-free diet from 10 days after mating. The question arises in the mechanism of action of the supplemental linoleic acid given to the progeny after weaning on PDHc activity. Any effect could obviously be either direct, due to variation in the PDHc environment, or could be secondary and due to the stimulation of lipoic acid synthesis needed for further activation of the lipoyl transacetylase and lipoamide dehydrogenase. ... 

Three different protein molecules are probably involved a more or less specific decarboxylase catalysing reaction 1, a lipoic acid reductase transacetylase catalysing reactions 2 and 3, and lipoamide-oxido-reductase catalysing reaction 4, the three proteins being combined in a multi-enzyme particle. In... 

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