ROLE OF SULFHYDRYL GROUPS IN CATALYTIC FUNCTION OF ISOCITRATE DEHYDROGENASE .I. REACTION WITH 5,5'-DITHIOBIS(2-NITROBENZOIC ACID)

The dehydrogenase and decarboxylase activities of the triphosphopyridine nucleotide dependent isocitrate dehydrogenase are both lost as a result of reaction with 5,5′-dithiobis(2-nitrobenzoicacid). Protection against inactivation is provided by isocitrate plus manganous sulfate and by reduced triphosphopyridine nucleotide plus manganous sulfate, but not by isocitrate, reduced pyridine nucleotide, or metal ion separately. A kinetic treatment is presented describing the initial velocity of an enzymatic reaction in the presence of a reversible inhibitor which reacts slowly but which competes with the substrate for the same or mutually exclusive sites. The rate of inactivation of isocitrate dehydrogenase by 5,5′-dithiobis(2-nitrobenzoic acid) in the presence of varying concentrations of isocitrate or reduced triphosphopyridine nucleotide and manganous sulfate is analyzed as a special case of this general mechanism. Alteration of the kinetic parameters of the enzyme is not responsible for the observed loss of activity of isocitrate dehydrogenase, since partially active preparations exhibit Michaelis constants and pH-rate profiles which are essentially identical with those of native enzyme. Incubation of the modified enzyme with mercaptoethanol results in a slow reactivation. Determination of the number of reactive sulfhydryl groups by the increase in absorbance at 412 mμ accompanying the release of 2-nitro-5-mercaptobenzoate from the reagent or by the enhanced absorbance at 323 mμ of the isolated modified enzyme yields a value of approximately five altered residues in the inactive enzyme. Only three SH groups react in the presence of isocitrate or reduced triphosphopyridine nucleotide and MnSO4, producing an active enzyme; a maximum of two sulfhydryl groups is thereby implicated in the loss of activity. Carboxymethylation of a single essential methionyl residue (Colman, R.F. (1968), J. Biol. Chem. 243, 2454) blocks one of the SH groups normally protected by isocitrate and manganous ion, demonstrating proximity between a sulfhydryl and a methionyl residue in the active site. © 1969, American Chemical Society. All rights reserved.