THE ACTIVITY OF LIVER ALCOHOL DEHYDROGENASE WITH NICOTINAMIDE-ADENINE DINUCLEOTIDE PHOSPHATE AS COENZYME

Abstract

The separa-tion of nucleotide impurities from commercial NADP preparations by chromatography is described. All the preparations studied contained 0.1-0. 2% of NAD. The activity of pure crystalline liver alcohol dehydrogenase with NADP as coenzyme has been confirmed. Initial-rate data are reported for the reaction at pH 6.0 and 7.0 with ethanol and acetaldehyde as substrates. With NADP and NADPH2 of high purity, the maximal specific rates were similar to those obtained with NAD and NADH2, but the Michaelis constants for the former coenzymes were much greater than those for the latter. The oxidation of etnanol by NADP is greatly inhibited by NADH2, and this accounts for low values of certain initial-rate parameters obtained with commercial NADP preparations containing NAD. The kinetics of the inhibition are consistent with competitive inhibition in a compulsory-order mechanism. Initial-rate data with NAD and NADPH2 do not conform to the requirements of the mechanism proposed by Theorell & Chance (1951), in contrast with results previously obtained with NAD and NADH2. The possibility that the deviations are due to competing nucleotide impurity in the oxidized coenzyme cannot be excluded. The data show that the enzyme reacts more slowly with, and has a smaller affinity for NADP and NADPH2 than NAD and NADH2. Phosphate behaves as a competitive inhibitor towards NADP.