Mechanistic study of the addition of pyruvate to NAD+ catalyzed by lactate dehydrogenase

Abstract

The binary complex of NAD+ and dogfish A4 lactate dehydrogenase reacts reversibly with pyruvate enol to produce an inactive, enzyme-adduct complex, in which the nicotinamide and pyruvate moieties are linked by means of a covalent bond. This process is examined in both the forward and reverse directions as a function of reactant and buffer concentrations at pH 7, under conditions where the enolization of pyruvate is at equilibrium, and the involvement of complexes with stoichiometry E.NAD, E.NAD.PyrE, and E.NAD.PyrK is defined. (The subscripts, E and K, indicate the enol and keto forms of pyruvate.) One pathway for formation of the adduct complex involves the prior formation of the E.NAD.PyrE complex from E.NAD and pyruvate enol; the alternative pathway involves formation of the same complex via the enolization of E.NAD.PyrK, a process that is catalyzed by an external (nonenzymic) base. The possible use of the adduct reaction as a model for the normal enzymic reaction is considered.