Enhancement of the Activity of Horse Liver Alcohol Dehydrogenase by Modification of Amino Groups at the Active Sites

Abstract

Abstract Reaction of horse liver alcohol dehydrogenase with imidoesters or cyanate at pH 8 significantly increases the activity of the enzyme, as assayed in high concentrations of NAD+ and ethanol at pH 9.0. Methyl picolinimidate activates the enzyme 19-fold and modifies about 50 of its 60 amino groups, as determined by spectral and amino acid analyses. When the active sites are protected with NAD+ and pyrazole (or NADH and isobutyramide) methyl picolinimidate activates only 2-fold, although most of the amino groups still react; after removal of the reagents by gel filtration, the partially substituted enzyme could be activated 11-fold more by methyl picolinimidate or 2-fold more by 14C-cyanate with the modification of about three amino groups per active site. A similar experiment with ethyl acetimidate in the first step and methyl picolinimidate in the second step gave similar results. Product inhibition studies show that the reactions catalyzed by both the native and picolinimidylated enzymes at pH 9.0 conform to the same mechanism, ordered bi bi. The modified enzyme has 12- to 53-fold larger Michaelis and inhibition constants for NADH and NAD+ and 12- and 30-fold larger turnover numbers. The rate-limiting step in either the forward or the reverse reaction with the native enzyme is the breakdown of the enzyme-coenzyme complex; the picolinimidylated dehydrogenase probably gives higher maximum velocities because the complexes dissociate faster. Picolinimidylation of the enzyme does not greatly affect the binding of AMP, ADP, or adenosine 5'-diphosphoribose, but markedly decreases the binding of NAD+, NADH, and 3-acetylpyridine adenine dinucleotide. The reactivities of the essential —SH groups and the zinc ions at the active sites of the enzyme are not affected by picolinimidylation. These results indicate that the amino groups that can be modified are not required for the catalytic activity of the enzyme and that there is probably at least one amino group near the binding site for the nicotinamide ring of the coenzyme.