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J. Biol. Chem., Vol. 280, Issue 34, 30349-30353, August 26, 2005

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Location of the Coenzyme Binding Site in the Porcine Mitochondrial NADP-dependent Isocitrate Dehydrogenase^*

Yu Chu Huang and Roberta F. Colman

From the Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716

The structure of crystalline porcine mitochondrial NADP-dependent isocitrate dehydrogenase (IDH) has been determined in complex with Mn²⁺-isocitrate. Based on structural alignment between this porcine enzyme and seven determined crystal structures of complexes of NADP with bacterial IDHs, Arg⁸³, Thr³¹¹, and Asn³²⁸ were chosen as targets for site-directed mutagenesis of porcine IDH. The circular dichroism spectra of purified wild-type and mutant enzymes are similar. The mutant enzymes exhibit little change in K_m for isocitrate or Mn²⁺, showing that these residues are not involved in substrate binding. In contrast, the Arg⁸³ mutants, Asn³²⁸ mutants, and T311A exhibit 3-20-fold increase in the . We propose that Arg⁸³ enhances NADP affinity by hydrogen bonding with the 3'-OH of the nicotinamide ribose, whereas Asn³²⁸ hydrogen bonds with N1 of adenine. The pH dependence of V_max for Arg⁸³ and Asn³²⁸ mutants is similar to that of wild-type enzyme, but for all the Thr³¹¹ mutants, pK_es is increased from 5.2 in the wild type to 6.0. We have previously attributed the pH dependence of V_max to the deprotonation of the metal-bound hydroxyl of isocitrate in the enzyme-substrate complex, prior to the transfer of a hydride from isocitrate to NADP's nicotinamide moiety. Thr³¹¹ interacts with the nicotinamide ribose and is the closest of the target amino acids to the nicotinamide ring. Distortion of the nicotinamide by Thr³¹¹ mutation will likely be transmitted to Mn²⁺-isocitrate resulting in an altered pK_es. Because porcine and human mitochondrial NADP-IDH have 95% sequence identity, these results should be applicable to the human enzyme.

Received for publication, May 27, 2005 , and in revised form, June 17, 2005.

^* This research was supported by National Institutes of Health Grant R01 HL67774. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716. Tel.: 302-831-2973; Fax: 302-831-6335; E-mail: rfcolman{at}chem.udel.edu.

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