Lipoyl Domain-based Mechanism for the Integrated Feedback Control of the Pyruvate Dehydrogenase Complex by Enhancement of Pyruvate Dehydrogenase Kinase Activity

Abstract

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 NH 2 -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 acetylCoA. As suggested by those results, the enhancement of kinase activity is very responsive to reductive acetylation with a half-maximal stimulation achieved with ϳ20% 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 revers...

Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases

“…Both PDKs and PDPs are bound to the lipoyl domains of E2 in PDC (Yang et al, 1998). The shortterm regulation of PDC activity is achieved through modulation of PDK activity (and correspondingly the level of PDC phosphorylation) through the changes in the status of the lipoyl groups from oxidized to reduced and acetylated forms (Ravindran et al, 1996). The longterm regulation of PDC is exerted at the transcriptional level by increased expression of PDK (leading to hyperphosphorylation of PDC) in response to different pathological conditions (diabetes, starvation).…”

Nematode pyruvate dehydrogenase kinases: role of the C-terminus in binding to the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex

“…Native and recombinant mammalian PDKs exist as dimers and seem to move between lipoyl domains without dissociation, as recently described by a ' hand-over-hand ' model [15]. This model predicts that each PDK homodimer should have two lipoyl-binding domains and suggests that an individual ' binding site ' might be sufficient for association with the PDC.…”

“…This observation is probably not surprising, given the markedly different intramitochondrial redox environments in which the PDKs are found. Stimulation by NADH and acetyl-CoA is dependent on the catalytic formation of acetyl dihydrolipoate by the PDC and is mediated by the E3-catalysed reduction and E2-catalysed acetylation of the lipoyl prosthetic group of E2 [15]. In mammalian PDCs, E3BP also contains an N-terminal lipoyl domain, although its role in the binding and regulation of the PDKs is unclear [35].…”

“…These results are in agreement with those reported for mammalian PDKs and suggest that the domains responsible for the binding of the nematode PDKs to the lipoyl domain of E2 are conserved, because both rAPDK and rCPDK bound the A. suum PDC. Previous workers also have demonstrated that PDKs are sensitive to the E3-catalysed reduction and E2-catalysed acetylation of the lipoyl groups of E2 [13][14][15]. Therefore to examine the effects of NADH and acetyl-CoA on the activity of the nematode rPDKs, the rPDKs were incubated with PDK-depleted PDC in NADH\ NAD + (1 : 200 or 3 : 1 molar ratios) or NADH\NAD + plus acetylCoA and then assayed for PDK activity, as described recently for the characterization of the mammalian PDK isoforms [9].…”

“…effects of NADH and acetyl-CoA on PDK activity are mediated by the degree of E3-catalysed reduction and E2-catalysed acetylation of the inner lipoyl domain of E2 [13][14][15], and each PDK isoform seems to exhibit a different sensitivity to NADH and acetyl-CoA [9]. The PDC has a key role in the anaerobic mitochondrial metabolism of the parasitic nematode Ascaris suum and is designed to function under the elevated intramitochondrial NADH\NAD + and acetyl-CoA\CoA ratios present during anaerobiosis [16,17].…”