Contribution to the knowledge of the role of SH-groups in the molecule of pea alcohol dehydrogenase

Abstract

p-Chloromercuribenzoate irreversibly inactivates alcohol dehydrogenase (ADH) isolated from germinating pea seeds. The reaction follows the first order kinetics. The inactivation of pea ADH is pH-dependent and is maximal at pH 9.0. NAD protects the enzyme from inactivation by p-chloromercuribenzoate; the higher the concentration of the coenzyme and the longer the period of incubation of NAD with the enzyme, the lower the degree of inactivation. Ethanol does not prevent the enzyme from inactivation. o-Phenanthroline in a concentration of 1 . 10-3 mol l-1 decreases the degree of inactivation of the enzyme by p-chloromercuribenzoate by 20%; imidazole is without effect on the reaction. Zn2+-ions in concentration of 1 . 10-5 mol l-1 also partly protect the enzyme from inactivation by p-chloromercuribenzoate. The results obtained show that the SH-groups sensitive to labeling with p-chloromercuribenzoate are localized in the active center of the enzyme, probably in the coenzyme-binding site. The protective action of Zn2+-ions and of o-phenanthroline against this inactivation confirms the assumption that the SH-group acts as a zinc ligand in the active center of the enzyme.