Kinetics of native and activated isozymes of horse liver alcohol dehydrogenase.

Abstract

The major isozymes of horse liver alcohol dehydrogenase (EC 1.1.1.1), EE, ES, and SS, have been separated by chromatography on phosphocellulose. Product inhibition studies showed that the kinetic behavior of EE and SS isozymes was consistent with the ordered BiBi mechanism. The different primary structures of the E and S subunits were expressed with higher Michaelis constants for ethanol and acetaldehyde and lower activity for the SS isozyme when compared with the EE isozyme. The differences for SS isozyme are reflections of slower rates of association and dissociation of coenzymes and slower rates of hydrogen transfer, not of affinities for the substrates. The contributions of each subunit in ES isozyme to the kinetic constants were not additive, indicating that the subunits may not act independently. Activation of the isozymes by amidination and alkylation suggested that lysine residues were present at the active sites of both E and S subunits. Kinetic studies indicated that isonicotinimidylation increased enzyme activity of the three isozymes by increasing the rates of dissociation of the enzyme-coenzyme complexes.