The kinetics of interconversion of intermediates of the reaction of pig muscle lactate dehydrogenase with oxidized nicotinamide–adenine dinucleotide and lactate

Abstract

Oxamate competes with pyruvate for the substrate binding site on the E^NADH complex of pig skeletal muscle lactate dehydrogenase. When this enzyme was mixed with saturating concentrations of NAD⁺ and lactate in a stopped-flow rapid-reaction spectrophotometer there was no transient accumulation of enzyme complexes with the reduced nucleotide. The steady-state rate of formation of free NADH was reached within the dead-time of the instrument (3ms). When oxamate was added to inhibit the steady state and to uncouple the equilibration: [Formula: see text] through the rapid formation of E^NADH_Oxamate, the rate of formation of E^NADH could be measured by observation of the first turnover. This pH-dependent transient is controlled by the rate of dissociation of pyruvate and the fraction of the enzyme in the form E^NADH_Pyruvate.