Ser95, Asn97, and Thr78 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 gamma-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 gamma-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 pK(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 gamma-carboxylate of isocitrate may position the substrate to promote a favorable lowering of the pK 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.
Kim
Tae-Kang
TK
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Colman
Roberta F
RF
D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D002384:Catalysis; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012694:Serine / Q000737:chemistry*; D013552:Swine; D013912:Threonine / Q000737:chemistry*
D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D001216:Asparagine; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)