Rapid intersite transfer of acetyl groups and movement of pyruvate dehydrogenase component in the kidney pyruvate dehydrogenase complex.

R.L Cate,T.E Roche,L.C Davis

doi:10.1016/s0021-9258(19)43864-7

Abstract

Two sites per subunit of the dihydrolipoyl transacetylase component of the kidney pyruvate dehydrogenase complex can be acetylated, and high levels of acetylation can be achieved under conditions in which only a few subunits of the pyruvate dehydrogenase component are functional (Cate, R. L., and Roche, T. E. (1979) J. Biol. Chem 254, 1659-1665). These obsewations suggest intersite transfer of acetyl groups between lipoyl moieties (two per subunit) of the dihydrolipoyl transacetylase component. We have used flowquench rapid reaction studies to evaluate whether a relay system functions to transfer acetyl groups rapidly between sites on the dihydrolipoyl transacetylase core. In complexes containing reduced levels of active pyruvate dehydrogenase component, four to seven acetyl groups per active pyruvate dehydrogenase component are introduced at a constant rate nearly equal, for the fraction of active enzyme, to the rate of the overall reaction catalyzed by the complex. The initial round of decarboxylation proceeds at a much faster rate than the incorporation of acetyl groups. Since both the first and second round of acetylation occurs at a constant rate equal to the rate of the overall reaction, reductive acetylation is the rate-limiting step in the overall reaction. Furthermore, if only one lipoyl moiety services an a/3 unit of the pyruvate dehydrogenase component, the above data indicate that there is subsequent interlipoyl transfer of acetyl groups which occurs at a faster rate than the reductive acetylation reaction.

Highlights

In complexes containing reducedlevels of active pyru- transfer of these groups (2, 3, 5). To evaluate whether such vate dehydrogenase component, four to seven acetyl transfers may be an essential part of the mechanism of the groups per active pyruvate dehydrogenase component complex, information is needed as to whether the transfers are introduced at a constant rate nearly equal, for the are rapid enough to contribute to the multistep catalysis of fraction of active enzyme, t o the rate of the overall the complex
Second round of acetylation occurs at a constant This suggested that interlipoyl transfer of acetyl groups may rate equal tothe rate of the overall reaction, reductive be kinetically important in this bacterial complex
The results indicate that atransfer of acetyl groups occurs in the kidney complex and is fast enough to be an important part of the catalytic mechanism

Summary

RESULTS

Dehydrogenase and dihydrolipoyl transacetylase, were prepared by Determination of the Number ofActive Pyruvate Dehydrothe method of Linn et al (13), except that pyruvate dehydrogenase was not crystallized. Interlipoyl Transfer of Acetyl Groups-Control samples tial rate was determined from the point of intersection proand samples inactivated to variouslevels of active pyruvate duced by a linear extrapolation of the initial phase and the dehydrogenase component by titration withTTPP were used linear portion of the second phase. This assumes that these to determine the rate of acetylation in quenched-flow rapid phases develop in a sequential manner for reasons discussed reaction experiments conducted in the absence of coenzyme below. It is not oossible to do this control with CoA which was present at 0.125 mM in the NAD-reduction assay

The time courses of acetylation by the various samples of

Transfers in MammPalyiraunvate

Findings

Transfers in Mammalian Pyruvate Dehydrogenase Complex