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J. Biol. Chem., Vol. 281, Issue 32, 23237-23245, August 11, 2006

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Determinants of the Dual Cofactor Specificity and Substrate Cooperativity of the Human Mitochondrial NAD(P)⁺-dependent Malic Enzyme

FUNCTIONAL ROLES OF GLUTAMINE 362^*

Ju-Yi Hsieh¹, Guang-Yaw Liu¹, Gu-Gang Chang^¶, and Hui-Chih Hung²

From the Department of Life Sciences, National Chung-Hsing University, Taichung 40227, the Institute of Immunology, Chung-Shan Medical University, Taichung 40201, and the ^¶Faculty of Life Sciences and the Institute of Biochemistry, National Yang-Ming University, Taipei 11221, Taiwan

The human mitochondrial NAD(P)⁺-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys³⁶² in the pigeon cytosolic NADP⁺-dependent malic enzyme has remarkable effects on the binding of NADP⁺ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln³⁶² in the transformation of cofactor specificity from NAD⁺ to NADP⁺ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD⁺ to NADP⁺. The K_m(NADP) and k_cat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in K_m(NADP)/K_m(NAD) and elevation of k_cat(NADP)/k_cat(NAD) were observed for the Q362K enzyme. Mutation of Gln³⁶² to Ala or Asn did not shift its cofactor preference. The K_m(NADP)/K_m(NAD) and k_cat(NADP)/k_cat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The G values for Q362A and Q362N with either NAD⁺ or NADP⁺ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln³⁶² to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln³⁶² to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln³⁶² mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD⁺ specificity. In this study, we provide clear evidence that the single mutation of Gln³⁶² to Lys in human m-NAD-ME changes it to an NADP⁺-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP⁺-specific.

Received for publication, April 11, 2006 , and in revised form, June 2, 2006.

^* This work was supported by Grant NSC-93-2311-B-005-018 (to H.-C. H.) from the National Science Council, Taiwan. 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.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Life Sciences, National Chung-Hsing University, 250, Kuo-Kuang Rd., Taichung 40227, Taiwan. Tel.: 886-4-2284-0416 (ext. 615); Fax: 886-4-2285-1856; E-mail: hchung{at}dragon.nchu.edu.tw.

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