Conceptual Error in Determination of NAD⁺-Malic Enzyme in Extracts Containing NAD⁺-Malic Dehydrogenase

Abstract

The typical procedure for determining NAD(+)-malic enzyme (EC 1.1.1.39) is to calculate the enzyme rate to be DeltaA(340)/Delta time after the endogenous NAD(+)-malic dehydrogenase (EC 1.1.1.37) catalyzed reaction has reached equilibrium. This ignores the equilibrium shift of oxaloacetic acid and NADH during the course of the NAD(+)-malic enzyme reaction and causes an error that varies depending on the reagent [malate], [NAD(+)], pH and final [NADH]. For a DeltaA(340) of 0.02, the error is about 80% and for a DeltaA(340) of 0.30, 20%. We develop this argument, give supportive data and present a simple method to circumvent the error.