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標題: Engineering of the Cofactor Specificities and Isoform-specific Inhibition of Malic Enzyme
作者: Hsieh, J.Y.
Hung, H.C.
關鍵字: ascaris-suum;functional roles;oxidative decarboxylases;crystal-structure;cdna cloning;binding-site;mechanism;purification;malate;nad
Project: Journal of Biological Chemistry
期刊/報告no:: Journal of Biological Chemistry, Volume 284, Issue 7, Page(s) 4536-4544.
Malic enzyme (ME) is a family of enzymes that catalyze a reversible oxidative decarboxylation of L-malate to pyruvate with simultaneous reduction of NAD(P)(+) to NAD(P)H. According to the cofactor specificity, the mammalian enzyme can be categorized into three isoforms. The cytosolic (c) and mitochondrial (m) NADP(+)-dependent MEs utilize NADP(+) as the cofactor. The mitochondrial NAD(P)(+)-dependent ME can use either NAD(+) or NADP(+) as the cofactor. In addition, the m-NAD(P)-ME isoform can be inhibited by ATP and allosterically activated by fumarate. In this study, we delineated the determinants for cofactor specificity and isoform-specific inhibition among the ME isoforms. Our data strongly suggest that residue 362 is the decisive factor determining cofactor preference. All the mutants containing Q362K (Q362K, K346S/Q362K, Y347K/Q362K, and K346S/Y347K/Q362K) have a larger k(cat,NADP) value compared with the k(cat,NAD) value, indicating that the enzyme has changed to use NADP(+) as the preferred cofactor. Furthermore, we suggest that Lys-346 in m-NAD(P)-ME is crucial for the isoform-specific ATP inhibition. The enzymes containing the K346S mutation (K346S, K346S/Y347K, K346S/Q362K, and K346S/Y347K/Q362K) are much less inhibited by ATP and have a larger K(i,ATP) value. Kinetic analysis also suggests that residue 347 functions in cofactor specificity. Here we demonstrate that the human K346S/Y347K/Q362K m-NAD(P)-ME has completely shifted its cofactor preference to become an NADP(+)-specific ME. In the triple mutant, Lys-362, Lys-347, and Ser-346 work together and function synergistically to increase the binding affinity for NADP(+).
ISSN: 0021-9258
DOI: 10.1074/jbc.M807008200
Appears in Collections:生命科學系所

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