The mammalian mitochondrial NADP-dependent isocitrate dehydrogenase is a citric acid cycle enzyme and
an important contributor to cellular defense against oxidative stress. The Mn2+-isocitrate complex of the
porcine enzyme was recently crystallized; its structure indicates that Ser95, Asn97, and Thr78 are within
hydrogen-bonding distance of the -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. Vmax
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 Km values for isocitrate and Mn2+. The results suggest that Thr78 and Ser95 function to strengthen the
enzyme’s affinity for Mn2+-isocitrate by hydrogen bonding to the -carboxylate of isocitrate. For the Asn97
mutants, the Km values are much less affected. The major change in the N97A mutant is the increase in pKa
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 Asn97 and the -carboxylate of isocitrate may position the
substrate to promote a favorable lowering of the pK of the enzyme–isocitrate complex. Thus, Thr78, Ser95,
and Asn97 perform important but distinguishable roles in catalysis by porcine NADP-specific isocitrate
dehydrogenase.