Oxamate competes with pyruvate for the substrate binding site on the ENADH 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 ENADHOxamate, the rate of formation of ENADH 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 ENADHPyruvate.

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