Function and regulation of mammalian pyruvate dehydrogenase complex. Acetylation, interlipoyl acetyl transfer, and migration of the pyruvate dehydrogenase component.

We have presented evidence that stimulation of pyruvate dehydrogenase, (PDH.) kinase activity by pyruvate or by acetyl-CoA is mediated through acetylation of lipoyl moieties (Cate, R. L., and Roche, T. E. (1978) J. Biol. Chem. 253,496-503). In accord with this indirect mechanism for the action of these effecters, we now find that the degree of stimulation of PDH, kinase increases with the level of acetylation with either [314C]pyruvate or [l-‘4C]acetyl-S-CoA as substrate until about 30 acetyl groups are incorporated per molecule of complex. Half-maximal stimulation is observed at about 5 to 6 acetyl residues incorporated. At ratios of effector to pyruvate dehydrogenase complex less than 40:1, virtually all the added effector (pyruvate or acetyl-CoA) is converted to protein-bound acetyl groups under the conditions selected for these experiments. These results clearly support a common mechanism involving acetylation of lipoyl moieties for stimulation of the activity of this converter enzyme. With both [3-14C]pyruvate and [l-‘4C]acetyl-S-CoA, about 100 mol of acid-stable acetyl residues are incorporated per mol of complex. Similar levels are also incorporated from pyruvate and acetyl-CoA into resolved dihydrolipoyl transacetylase. Since there are only 60 dihydrolipoyl transacetylase subunits/molecule of complex, more than one lipoyl moiety is acetylated per transacetylase subunit. High levels of acetylation with pyruvate are achieved under conditions in which only a few pyruvate dehydrogenase subunits are functional. Acetyl transfer between lipoyl moieties and interchange of the pyruvate dehydrogenase component among sites on the dihydrolipoyl transacetylase can contribute to acetylation under these conditions. The rate of acetylation due to movement of pyruvate dehydrogenase subunits is estimated and is not large enough to contribute to steady state catalysis by the pyruvate dehydrogenase complex. However, we estimate that movement of the pyruvate dehydrogenase component is fast enough to participate in, and possibly be an essential step for, the interconversion of PDH, by a fixed PDH, kinase.