title: Engineering of the cofactor specificities and isoform-specific inhibition of malic enzyme.
abstract: 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(+).
authors.affiliation: Department of Life Sciences and Institute of Bioinformatics, National Chung-Hsing University, Taichung 40227, Taiwan.
authors.forename: Ju-Yi
authors.initials: JY
authors.lastname: Hsieh
authors.forename: Hui-Chih
authors.initials: HC
authors.lastname: Hung
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D003653:Decarboxylation / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D009243:NAD / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000502:physiology; D020836:Protein Structure, Quaternary / Q000502:physiology
chemical_list: D007527:Isoenzymes; D024101:Mitochondrial Proteins; D009243:NAD; D009249:NADP; D008291:Malate Dehydrogenase