| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 96 === Cytosolic NADP+-dependent malic enzyme (c-NADP-ME) catalyzes a reversible oxidative decarboxylation converting L-malate into CO2 and pyruvate, concomitant with reduction of NADP+ to NADPH. Divalent metal ion (Mn2+ or Mg2+) is required for catalysis. According to the previous studies, mitochondrial malic enzyme (m-NAD-ME) is considered as an allosteric enzyme with fumarate as an activator and ATP as an inhibitor. In this study, we created a series of multiple mutations based on the sequence alignments to build a fumarate binding site for human c-NADP-ME. The optimal activating effect by fumarate was observed in the quadruple mutant (S57K/N59E/E73K/S102D). Our kinetic data clearly indicate that substitution of Ser57, Asn59, Glu73, and Ser102 is helpful in creation of fumarate binding site. In addition, previous kinetic studies have suggested that Ser346, Lys347, and Lys362 might be responsible for NADP+ selectivity. We further delineated the relationships of these three residues for the cofactor selectivity in human c-NADP-ME by creating three single mutants (S346K, K347Y, K362Q), three double mutants (S346K/K347Y, S346K/K362Q, K347Y/ K362Q), and one triple mutant (S346K/K347Y/K362Q). The cofactor preference shift from NADP+ to NAD+ was accomplished with the triple mutant, owing to the Km(NADP) and Km(NAD) values of the triple mutant were increased by 4822-fold and decreased by about 2-fold, respectively, compared with those of wild-type enzyme. Besides, the kcat(NAD) of triple mutant enzyme increased to a similar level of kcat(NADP) of wild-type enzyme. Thus, based on our kinetic data, we suggested that the balance of electric charge of these mutant enzymes might result in differential ATP inhibition by utilizing NAD+ or NADP+ as cofactor.
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