Ligands of the Mn2+ Bound to Porcine Mitochondrial NADP-Dependent Isocitrate Dehydrogenase, as Assessed by Mutagenesis

Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase requires a divalent metal ion for catalysis, and metal-isocitrate is its preferred substrate. On the basis of the crystal structure of the enzyme−Mn2+−isocitrate complex, Asp252, Asp275, and Asp279 were selected as targets for site-directed mutagenesis to evaluate the roles of these residues as ligands of the metal ion. The circular dichroism spectra of the purified mutant enzymes are similar to that of wild-type enzyme indicating there are no appreciable conformational changes. The K m values for isocitrate and for Mn2+ are increased in the asparagine and histidine mutants at positions 252 and 275; while for cysteine mutants at the same positions, the K m's are not changed appreciably. Mutants at position 279 exhibit only a small change in K m for isocitrate. These results indicate that Asp252 and Asp275 are ligands of enzyme-bound Mn2+and influence the binding of Mn2+−isocitrate. Cysteine is an acceptable substitute for aspartate as a ligand of Mn2+. The pK aes's of D252C and D275C enzymes are similar to that of the wild-type enzyme (about 5.2), while the pK aes of D279C is a little lower (about 4.7). These findings suggest that the V max's of the D252C, D275C, and D279C enzymes depend on the ionizable form of the same group as in wild-type enzyme and neither Asp252, Asp275, nor Asp279 acts as the general base in the enzymatic reaction. For wild-type enzyme, the pK aes varies with the metal ion used with Mg2+ > Cd2+ > Mn2+ > Co2+, similar to the order of the pK's for these four metal-bound waters. We therefore attribute the pH dependence of V max to the deprotonation of the metal-coordinated hydroxyl group of isocitrate bound to isocitrate dehydrogenase.