SLOW-BINDING INHIBITION OF THE ESCHERICHIA-COLI PYRUVATE-DEHYDROGENASE MULTIENZYME COMPLEX BY ACETYLPHOSPHINATE

The pyruvate analogue acetylphosphinate (CH3-CO-P02H2) inhibits the pyruvate dehydrogenase component (E1) of the Escherichia coli pyruvate dehydrogenase multienzyme complex in a time-dependent process with biphasic reaction kinetics. The formation of an initial, rapidly reversible enzyme-inhibitor complex (EI) with an apparent Ki of 0.12 ± 0.025 μM is followed by the conversion to a tighter complex (EI*) at a maximal rate of K3 = 0.87 ± 0.34 min−1. The inhibition is reversible (dissociation rate constant k4 = 0.038 ± 0.002 min−1), requires the presence of the cofactors thiamin pyrophosphate and Mg2+, and is competitive with regard to pyruvate. The microscopic rate constants give a value of 5 nM for the overall dissociation constant [Ki*= [E][I]/([EI] + [EI*]) = Kik4/(k3 + k4)] compared with values of 10 and 3.5 nM obtained by steady-state methods. Thus acetylphosphinate binds by 5 orders of magnitude more tightly to pyruvate dehydrogenase than does pyruvate (Km = 0.35 mM). Acetylphosphinate also affects the pyruvate dehydrogenase complex fluorescence when excited at 290 nm in a time-dependent manner with a maximal rate constant of 0.99 min−1, suggesting a conformational change in the enzyme complex as the slow step in conversion of EI to EI* (K3). All these features taken together suggest that the interaction of the pyruvate dehydrogenase with acetylphosphinate involves the formation of a thiamin pyrophosphate-acetylphosphinate adduct that resembles the normal reaction intermediate, 2-(l-carboxy-l-hydroxyethyl)thiamin pyrophosphate (α-lactylthiamin pyrophosphate). © 1990 American Chemical Society. All rights reserved.