EFFECT OP PHOSPHORYLATION ON THE CATALYTIC FUNCTION OF MUSCULAR PYRUVATE-DEHYDROGENASE COMPLEX

Phosphorylation of pyruvate dehydrogenase complex (PDC) isolated from pigeon m. pectoralis by endogenous ATP-dependent protein kinase inhibi ts substrate conversion in oxidoreductase and non-oxidative reactions that involve the decay of pyruvate in the absence of coenzyme A or nic otinamide adenine dinucleotide. Circular dichroic spectra were used to monitor the formation and breakdown of the complex with charge transf er between component E1 of PDC and thiamine pyrophosphate during catal ysis. The technique allowed observation of a phosphorylation-inhibitab le catalytic step. Phosphorylation of PDC reduces its affinity for coe nzyme, does not preclude formation of holo-E1, but prevents it from in teracting with pyruvate. Phosphorylated or dephosphorylated PDC intera cts with 2-hydroxyethyl thiamine pyrophosphate in a similar way involv ing half of their active sites; all active sites of E1 work in the pre sence of deacylating agents (coenzyme A and dithiothreitol). The data suggest that phosphorylated E1 preserves the regimen of alternating op eration of active sites in reductive acetylation of the acceptor subst rate.