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Ser⁹⁵, Asn⁹⁷, and Thr⁷⁸ are important for the catalytic function of porcine NADP-dependent isocitrate dehydrogenase

Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA

(RECEIVED August 31, 2004; FINAL REVISION September 27, 2004; ACCEPTED September 28, 2004)

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 muta-genesis 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 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 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 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.

Keywords: isocitrate dehydrogenase; citric acid cycle; dehydrogenase

Abbreviations: PCR, polymerase chain reaction • IPTG, isopropylthio--D-galactopyranoside • TEMED, N,N,N',N'-tetramethylethylenediamine • SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate • IDH, isocitrate dehydrogenase

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041091805.

Reprint requests to: Roberta F. Colman, Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA; e-mail: rfcolman{at}udel.edu; fax: (302) 831-6335.

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				Y. C. Huang and R. F. Colman Location of the Coenzyme Binding Site in the Porcine Mitochondrial NADP-dependent Isocitrate Dehydrogenase J. Biol. Chem., August 26, 2005; 280(34): 30349 - 30353. [Abstract] [Full Text] [PDF]