Functional Groups of Diphosphopyridine Nucleotide-linked Isocitrate Dehydrogenase from Bovine Heart

Abstract DPN-linked isocitrate dehydrogenase from bovine heart contains 6 half-cystine residues per subunit of molecular weight of 42,000. All of these residues are present as cysteine since six sulfhydryl groups per subunit can be modified by 5',5'-dithiobis(2-nitrobenzoate) (DTNB). Spectrophotometric measurements indicate that the modification of three thiol groups which react preferentially with DTNB is directly proportional to loss of activity, up to 70% inactivation. 2-Mercaptoethanol and dithiothreitol completely restored activity of enzyme preparations where less than two sulfhydryl groups per subunit had been modified by DTNB, but less than 10% reversal was obtained when more than four sulfhydryl groups had been modified. No reversal with Na2SO3 and only partial reversal with KCN is obtained, even though both reagents release 2-nitro-5-mercaptobenzoate in stoichiometric amounts from the modified enzyme. Manganous isocitrate and, to a lesser extent, DPN+ and DPNH protect the enzyme against inactivation by DTNB. In the presence of 0.8 m (NH4)2SO4, one sulfhydryl group per subunit can be modified by DTNB and in the presence of manganous isocitrate, one additional group can be modified with full retention of activity. These results suggest that only one of the three reactive sulfhydryl groups is required for activity. The pH inactivation profile with DTNB shows that the activity-related thiol group has a pKa of 7.6. Increasing electrolyte concentration does not affect the rate of enzyme inactivation by DTNB; however, it does increase the reactivity of a sulfhydryl group which is not required for activity and decreases the protection given by manganous isocitrate. These results suggest that high salt concentrations interfere with binding of manganous isocitrate to the enzyme, possibly at an active center amino group (Fan, C. C., and Plaut, G. W. E. (1974) Biochemistry 13, 45–59). Lowering the temperature of the preincubation mixture from 23 to 0° decreases the rate of inactivation by DTNB by about 50% and almost abolishes the protection given by manganous isocitrate. In addition to DTNB, the enzyme is inactivated by p-chloromercuribenzene sulfonate, N-ethylmaleimide, iodoacetate, and iodoacetamide. Manganous isocitrate partially protects against inactivation by these sulfhydryl group modifying agents.