13C nuclear magnetic resonance study of the pyruvate dehydrogenase-catalyzed acetylation of dihydrolipoamide.

T P O'Connor,T E Roche,J V Paukstelis

doi:10.1016/s0021-9258(19)81081-5

Abstract

The dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex catalyzes a reversible reaction between acetyl-CoA and dihydrolipoamide that results in the formation of S-acetyldihydrolipoamide. We have used 13C nuclear magnetic resonance to investigate this reaction using exogenous forms of dihydrolipoamide in place of the protein-bound substrate. With substrate levels of dihydrolipoamide and enzymatically generated [1-13C]acetyl-CoA, both 6-S-[1-13C]acetyl- and 8-S-[1-13C]acetyldihydrolipoamide were formed in the transacetylation reaction and both species participated in the reverse reaction to yield [1-13C]acetyl-CoA and free dihydrolipoamide. The 8-S-acetyl derivative was the principal product. It is suggested that acetylation of both the 6- and 8-thiols of dihydrolipoamide results as a consequence of intramolecular migration following acetylation at a single site. After longer periods of reaction, some 6,8-S,S-[1-13C]diacetyldihydrolipoamide also accumulated. We have also found that [1-13C]acetyl-CoA reacts slowly with dihydrolipoamide in a nonenzymatic reaction to yield the two monoacetylated and some diacetylated derivative. In the reverse reaction catalyzed by the dihydrolipoyl transacetylase, it was clear that monoacetyl derivatives were depleted much more rapidly than the diacetyl derivatives, although we could not quantitate the change in the low concentration of the diacetyl derivative.

Highlights

It is suggested that acetylation of both the 6-and 8-thiols of dihydrolipoamide results as a consequence of intramolecular migration following acetylation at a single site
Reactions A and B are catalyzed by the pyruvate dehydrogenase component ( E, )and result in the acetylationof lipoic acid residues (Lip-&) covalently bound to the dihydrolipoyl transacetylase core component of the complex (E2)P. roteinbound S-acetyldihydrolipoamide is formed and reacts with CoA, in a dihydrolipoyl transacetylase-catalyzed reaction, to longer periods of reaction, some 6,8-S,S-[l-’3C]diacetyl- form acetyl-coAand dihydrolipoamide (Reaction C)
In the reverse reaction catalyzed by the dihydrolipoyl transacetylase, it was clear that monoacetyl derivatives were depleted much more rapidly than thediacetyl derivatives, we could not quantitate the change in the low concentration of the diacetyl derivative

Summary

EXPERIMENTA LP ROCEDURES

Materials-Imidazole (grade 111), 3-(N-morpholino)propanesulfonic acid, NAD’ (grade III), potassium pyruvate,dithiothreitol, thiaminpyrophosphate, sodium borohydride, and lipoamide (6, 8tbioctic acid amide) were purchased from Sigma. A unit of enzyme activity is 1pmol/min. Assays-The activity of the pyruvate dehydrogenase complex was measured by the procedures of Linn et al (7). Assay of the nonenzymatic reaction between [l-14C]acetyl-CoAand dihydrolipoamide was based on the method of Buttenvorth et al (2). Reaction was initiated by addition of [l-'4C]acetyl-CoA (1.0 mM) to a solution of dihydrolipoamide (10.0 mM) in 0.05 M Mops buffer (pH 7.5). The efficiency of extraction of the labeled product was approximately 85%;the reaction was blanked against samples lacking dihydrolipoamide. For analysis of this nonenzymatic reaction by NMR, [l-'3C]acetyl-CoA was prepared enzymatically, followed by precipitation of the protein with trichloroacetic acid, removal of the precipitate by centrifugation and reneutralization to pH 7.5 with KOH. Chemical shifts in aqueous buffers were very similar to those in CDC13

RESULTS

Dihydrolipoyl Transacetylase Acetylation of Dihydrolipoamide

Findings

DISCUSSION