Abstract
Crystalline preparations of the glutamic dehydrogenase of beef liver contain from 2 to 4 gm. atoms of zinc per mole of enzyme protein (1). The dependence of the catalytic activity of glutamic dehydrogenase on the metal is inferred from (a) the concomitant increase of zinc to protein and activity to protein ratios during its crystallization and (b) the inhibition of enzyme activity by agents which form stable complexes with zinc ions in aqueous solutions. In this report the inhibition of glutamic dehydrogenase by metal-binding agents, including 1, IO-phenanthroline, is shown to depend on the ability of these agents to form a complex with zinc ions and to be qualitatively similar to that observed with yeast alcohol dehydrogenase, another zinc metallobehydrogenase. A mechanism for the inhibition of glutamic dehydrogenase by metal-binding agents similar to that observed for another zinc metallodehydrogenase, yeast alcohol dehydrogenase, (2, 3) is proposed, although glutamic dehydrogenase differs from alcohol dehydrogenase both with respect to the reaction it catalyzes and its protein size and structure.
Highlights
Injluence of Metal-binding Agents on Enzymatic Activity-The direct addition of sodium sulfide (1 x 10” M), sodium azide (3 X 10-l M), OP (3 X 1O-3 M), thiourea (3 X 10-l M), diethyldithiocarbamate (3 X 10m2 M), Shydroxyquinoline (3 X low3 M), cupferron (3 X 10e3 M), EDTA (1.7 X 10-l M), Zincon (6 X 1O-5 M), and thyroxine (8 X 1O-4 M) to the reaction cuvette all reduce the enzymatic activity of glutamic dehydrogenase [1]
Beef liver glutamic dehydrogenase is inhibited by metal-binding agents, both when these are present in the enzyme mixture and when preincubated with the enzyme before assay
Beef liver glutamic dehydrogenase is inhibited by metal-binding agents including sodium sulfide, sodium azide, 1, lo-phenanthroline, diethyldithiocarbamate
Summary
Injluence of Metal-binding Agents on Enzymatic Activity-The direct addition of sodium sulfide (1 x 10” M), sodium azide (3 X 10-l M), OP (3 X 1O-3 M), thiourea (3 X 10-l M), diethyldithiocarbamate (3 X 10m2 M), Shydroxyquinoline (3 X low M), cupferron (3 X 10e3 M), EDTA (1.7 X 10-l M), Zincon (6 X 1O-5 M), and thyroxine (8 X 1O-4 M) to the reaction cuvette all reduce the enzymatic activity of glutamic dehydrogenase [1]. Prevention of OP Inhibition by Metal Ions-It has been shown previously that the instantaneous inhibition of glutamic dehydrogenase is reversed by the addition of zinc chloride [1]. Zinc ions, which form strong complexes with OP, prevent the inhibition of the enzyme by the reagent. Increasing concentrations of zinc chloride added to OP before the addition of enzyme to the reaction mixture progressively prevent inactivation. Inactivation of the enzyme is complete when the ratio of [Zn++]:[OP] becomes 1 to 1
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