Ser 95 , Asn 97 , and Thr 78 are important for the catalytic function of porcine NADP‐dependent isocitrate dehydrogenase

The mammalian mitochondrial NADP‐dependent isocitrate dehydrogenase is a citric acid cycle enzyme and an important contributor to cellular defense against oxidative stress. The Mn 2+ ‐isocitrate complex of the porcine enzyme was recently crystallized; its structure indicates that Ser 95 , Asn 97 , and Thr 78 are within hydrogen‐bonding distance of the γ‐carboxylate of enzyme‐bound isocitrate. We used site‐directed mutagenesis to replace each of these residues by Ala and Asp. The wild‐type and mutant enzymes were expressed in Escherichia coli and purified to homogeneity. All the enzymes retain their native dimeric structures and secondary structures as monitored by native gel electrophoresis and circular dichroism, respectively. V max of the three alanine mutants is decreased to 24%–38% that of wild‐type enzyme, with further decreases in the aspartate mutants. For T78A and S95A mutants, the major changes are the 10‐ to 100‐fold increase in the K m values for isocitrate and Mn 2+ . The results suggest that Thr 78 and Ser 95 function to strengthen the enzyme's affinity for Mn 2+ ‐isocitrate by hydrogen bonding to the γ‐carboxylate of isocitrate. For the Asn 97 mutants, the K m values are much less affected. The major change in the N97A mutant is the increase in p K a of the ionizable metal‐liganded hydroxyl of enzyme‐bound isocitrate from 5.23 in wild type to 6.23 in the mutant enzyme. The hydrogen bond between Asn 97 and the γ‐carboxylate of isocitrate may position the substrate to promote a favorable lowering of the p K of the enzyme–isocitrate complex. Thus, Thr 78 , Ser 95 , and Asn 97 perform important but distinguishable roles in catalysis by porcine NADP‐specific isocitrate dehydrogenase.