Characterization and kinetics of native and chemically acitvated human liver alcohol dehydrogenases.

Abstract

Acetimidylation of the amino groups of alcohol dehydrogenase from human and horse liver yields several modified enzyme forms, which differ in electrophoretic mobility and can be separated by ion exchange chromatography, but which are similar in kinetic characteristics. The acetimidylated, as well as the methylated, enzymes from human livers of the normal phenotype have increased activity and larger Michaelis and inhibition constants. These results suggest that the human enzyme has amino groups at the active sites, as was shown previously for the horse enzyme. The variant subunit occuring in the enzyme isolated from atypical human livers does not seem to be activated by acetimidylation, which may indicate that substitution of proline for Ala-230 or modifiction of Lys-228 is sufficient to fully activate the enzyme. Results of product inhibition studies of native and modified human enzymes are consistent with an Ordered Bi Bi mechanism. However, the major isoenzyme of native human liver alcohol, dehydrogenase exhibits nonlinear kinetics over a wide range of ethanol concentrations. This result may indicate that subunits with different kinetic characteristics are present or that there is negative cooperativity between subunits. After chemical modification, the kinetic patterns become linear, suggesting that the mechanism is altered.