LIPOYL DOMAIN-BASED MECHANISM FOR THE INTEGRATED FEEDBACK-CONTROL OF THE PYRUVATE-DEHYDROGENASE COMPLEX BY ENHANCEMENT OF PYRUVATE-DEHYDROGENASE KINASE-ACTIVITY

To conserve carbohydrate reserves, the reaction of the pyruvate dehydr ogenase complex (PDC) must be down-regulated when the citric acid cycl e is provided sufficient acetyl-CoA., PDC activity is reduced primaril y through increased phosphorylation of its pyruvate dehydrogenase (El) component due to El kinase activity being markedly enhanced by elevat ed intramitochondrial NADH:NAD(+) and acetyl-CoA:CoA ratios, A mechani sm is evaluated in which enhanced kinase activity is facilitated by th e build up of the reduced and acetylated forms of the lipoyl moieties of the dihydrolipoyl acetyltransferase (E2) component through using NA DH and acetyl-CoA in the reverse of the downstream reactions of the co mplex, Using a peptide substrate, kinase activity was stimulated by th ese products, ruling out the possibility kinase activity is increased due to changes in the reaction state of its substrate, El (thiamin pyr ophosphate), Each E2 subunit contains two lipoyl domains, an NH2-termi nal (L1) and the inward lipoyl domain (L2), which were individually pr oduced in fully lipoylated forms by recombinant techniques, Although r eduction and acetylation of the L1 domain or free lipoamide increased kinase activity, those modifications of the lipoate of the kinase-bind ing L2 domain gave much greater enhancements of kinase activity, The l arge stimulation of the kinase generated by acetyl-CoA only occurred u pon addition of the transacetylase-catalyzing (lipoyl domain-free) inn er core portion of E2 plus a reduced lipoate source, affirming that ac etylation of this prosthetic group is an essential mechanistic step fo r acetyl-CoA enhancing kinase activity. Similarly, the lesser stimulat ion of kinase activity by just NADH required a lipoate source, support ing the need for lipoate reduction by E3 catalysis. Complete enzymatic delipoylation of PDC, the E2-kinase subcomplex, or recombinant L2 abo lished the stimulatory effects of NADH and acetyl-CoA. Retention of a small portion of PDC lipoates lowered kinase activity but allowed stim ulation of this residual kinase activity by these products, Reintroduc tion of lipoyl moieties, using lipoyl protein ligase, restored the cap acity of the E2 core to support high kinase activity along with stimul ation of that activity up to 3-fold by NADH and acetyl-CoA. As suggest ed by those results, the enhancement of kinase activity is very respon sive to reductive acetylation with a half-maximal stimulation achieved with similar to 20% of free L2 acetylated and, from an analysis of pr evious results, with acetylation of only 3-6 of the 60 L2 domains in i ntact PDC. Based on these findings, we suggest that kinase stimulation results from modification of the lipoate of an L2 domain that becomes specifically engaged in binding the kinase, In conclusion, kinase act ivity is attenuated through a substantial range in response to modest changes in the proportion of oxidized, reduced, and acetylated lipoyl moieties of the L2 domain of E2 produced by fluctuations in the NADH: NAD(+) and acetyl-CoA:CoA ratios as translated by the rapid and revers ible E3 and E2 reactions.