Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/38144
標題: Determinants of the dual cofactor specificity and substrate cooperativity of the human mitochondrial NAD(P)(+)-dependent malic enzyme - Functional roles of glutamine 362
作者: Hsieh, J.Y.
洪慧芝
Liu, G.Y.
Chang, G.G.
Hung, H.C.
關鍵字: ascaris-suum;oxidative decarboxylases;regulatory properties;catalytic;mechanism;crystal-structure;adrenal-cortex;cdna cloning;rat-liver;nad;purification
Project: Journal of Biological Chemistry
期刊/報告no:: Journal of Biological Chemistry, Volume 281, Issue 32, Page(s) 23237-23245.
摘要: 
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 Lys(362) 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 Gln(362) 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 Km(NADP)/Km(NAD) and elevation of k(cat(NADP))/k(cat(NAD)) were observed for the Q362K enzyme. Mutation of Gln(362) 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 Delta 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 Gln(362) to Lys because the sigmoidal phenomenon appearing in the wildtype enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln(362) 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 Gln(362) mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD(+) specificity. In this study, we provide clear evidence that the single mutation of Gln(362) 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.
URI: http://hdl.handle.net/11455/38144
ISSN: 0021-9258
DOI: 10.1074/jbc.M603451200
Appears in Collections:生命科學系所

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