Cysteine in the manganous-isocitrate binding site of pig heart TPN-specific isocitrate dehydrogenase: I. Kinetics of chemical modification and properties of thiocyano enzyme

Pig heart TPN-dependent isocitrate dehydrogenase is inactivated by reaction with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB). The dependence of the rate constant for inactivation on the reagent concentration is nonlinear, and can be analyzed in terms of the existence of two mechanisms for reaction with the enzyme, one involving reversible binding prior to inactivation and the other a bimolecular reaction. Cyanide reacts with the inactive modified enzyme to yield thiocyano-isocitrate dehydrogenase without increasing the catalytic activity; this result suggests that inactivation by DTNB is not due to steric hindrance by the bulky thionitrobenzoate group bound to the enzyme. The inactive thiocyano enzyme binds manganous ion normally. In contrast to its effect on native enzyme, however, isocitrate does not strengthen the binding of Mn²⁺ to the thiocyano enzyme; the tightened binding of manganous-isocitrate may be critical for the catalytic activity of the enzyme. Protection against inactivation by DTNB is provided by isocitrate plus the activator, manganous ion, or the competitive inhibitor, calcium ion. The concerted inhibitors oxalacetate and glyoxylate, when present together with Mn²⁺ and TPN, also protect against loss of activity. A marked decrease in the inactivation rate constant to a finite limiting value is caused by saturating concentrations of TPNH and Mn²⁺, indicating that these ligands do not bind directly at the sites attacked by DTNB. The number of cysteine residues which react with DTNB concomitant with inactivation depends on the ligands present in the reaction mixture. In all cases, the equivalent of one -SH reacts without affecting activity. In the presence of Mn²⁺ and α-ketoglutarate, which do not appreciably affect the inactivation rate, loss of activity is proportional to reaction with two -SH groups. These results suggest that the integrity of a maximum of two cysteine residues is essential for the function of the pig heart isocitrate dehydrogenase, and that at least one cysteine residue may be located within the manganous-isocitrate binding site.