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| - Pyruvate Dehydrogenase Kinase Isoform 2 Activity Stimulated by Speeding Up theRate of Dissociation of ADP
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| Abstract
| - Pyruvate dehydrogenase kinase 2 (PDK2) activity is stimulated by NADH and NADH plusacetyl-CoA via the reduction and reductive acetylation of the lipoyl groups of the dihydrolipoylacetyltransferase (E2) component. Elevated K+ and Cl- were needed for significant stimulation. Stimulationsubstantially increased both kcat and the Km for ATP; the fractional stimulation increased with the level ofATP. With an E2 structure lacking the pyruvate dehydrogenase (E1) binding domain, stimulation of PDK2was retained, the Km for E1 decreased, and the equilibrium dissociation constant for ATP increased butremained much lower than the Km for ATP. Stimulation of PDK2 activity greatly reduced the fraction ofbound ADP. These results fit an ordered reaction mechanism with ATP binding before E1 and stimulationincreasing the rate of dissociation of ADP. Conversion of all of the lipoyl groups in the E2 60mer to theoxidized form (E2ox) greatly reduced kcat and the Km of PDK2 for ATP. Retention over an extendedperiod of time of a low portion of reduced lipoyl groups maintains E2 in a state that supported muchhigher PDK2 activity than short-term (5 min) reduction of a large portion of lipoyl groups of E2ox, butreduction of E2ox produced a larger fold stimulation. Reduction and to a greater extent reductive acetylationincreased PDK2 binding to E2; conversion to E2ox did not significantly hinder binding. We suggest thatpassing even limited reducing equivalents among lipoyl groups maintains E2 lipoyl domains in aconformation that aids kinase function.
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