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 dehydrogenase complex (PDC) must be down-regulated when the citric acid cycle is provided sufficient acetyl-CoA. PDC activity is reduced primarily through increased phosphorylation of its pyruvate dehydrogenase (E1) component due to E1 kinase activity being markedly enhanced by elevated intramitochondrial NADH:NAD+ and acetyl-CoA:CoA ratios. A mechanism is evaluated in which enhanced kinase activity is facilitated by the build-up of the reduced and acetylated forms of the lipoyl moieties of the dihydrolipoyl acetyltransferase (E2) component through using NADH and acetyl-CoA in the reverse of the downstream reactions of the complex. Using a peptide substrate, kinase activity was stimulated by these products, ruling out the possibility kinase activity is increased due to changes in the reaction state of its substrate, E1 (thiamin pyrophosphate). Each E2 subunit contains two lipoyl domains, an NH2-terminal (L1) and the inward lipoyl domain (L2), which were individually produced in fully lipoylated forms by recombinant techniques. Although reduction and acetylation of the L1 domain or free lipoamide increased kinase activity, those modifications of the lipoate of the kinase-binding L2 domain gave much greater enhancements of kinase activity. The large stimulation of the kinase generated by acetyl-CoA only occurred upon addition of the transacetylase-catalyzing (lipoyl domain-free) inner core portion of E2 plus a reduced lipoate source, affirming that acetylation of this prosthetic group is an essential mechanistic step for acetyl-CoA enhancing kinase activity. Similarly, the lesser stimulation of kinase activity by just NADH required a lipoate source, supporting the need for lipoate reduction by E3 catalysis. Complete enzymatic delipoylation of PDC, the E2-kinase subcomplex, or recombinant L2 abolished the stimulatory effects of NADH and acetyl-CoA. Retention of a small portion of PDC lipoates lowered kinase activity but allowed stimulation of this residual kinase activity by these products. Reintroduction of lipoyl moieties, using lipoyl protein ligase, restored the capacity of the E2 core to support high kinase activity along with stimulation of that activity up to 3-fold by NADH and acetyl-CoA. As suggested by those results, the enhancement of kinase activity is very responsive to reductive acetylation with a half-maximal stimulation achieved with approximately 20% of free L2 acetylated and, from an analysis of previous results, with acetylation of only 3-6 of the 60 L2 domains in intact 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 activity 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 reversible E3 and E2 reactions.
Ravindran
S
S
Department of Biochemistry, Kansas State University, Manhattan 66506, USA.
Radke
G A
GA
Guest
J R
JR
Roche
T E
TE
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism; D004789:Enzyme Activation; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005246:Feedback; D009243:NAD / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D013379:Substrate Specificity
D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D000105:Acetyl Coenzyme A; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D000581:Amidohydrolases; C023940:lipoamidase