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|標題:||Determinants of Nucleotide-Binding Selectivity of Malic Enzyme||作者:||Hsieh, J.Y.
|關鍵字:||nicotinamide adenine-dinucleotide;functional roles;oxidative;decarboxylases;catalytic mechanism;crystal-structure;cdna cloning;atp;purification;expression;pigeon||Project:||Plos One||期刊/報告no：:||Plos One, Volume 6, Issue 9.||摘要:||
Malic enzymes have high cofactor selectivity. An isoform-specific distribution of residues 314, 346, 347 and 362 implies that they may play key roles in determining the cofactor specificity. Currently, Glu314, Ser346, Lys347 and Lys362 in human c-NADP-ME were changed to the corresponding residues of human m-NAD(P)-ME (Glu, Lys, Tyr and Gln, respectively) or Ascaris suum m-NAD-ME (Ala, Ile, Asp and His, respectively). Kinetic data demonstrated that the S346K/K347Y/K362Q c-NADP-ME was transformed into a debilitated NAD(+)-utilizing enzyme, as shown by a severe decrease in catalytic efficiency using NADP(+) as the cofactor without a significant increase in catalysis using NAD(+) as the cofactor. However, the S346K/K347Y/K362H enzyme displayed an enhanced value for k(cat,NAD), suggesting that His at residue 362 may be more beneficial than Gln for NAD(+) binding. Furthermore, the S3461/K347D/K362H mutant had a very large K(m,NADP) value compared to other mutants, suggesting that this mutant exclusively utilizes NAD(+) as its cofactor. Since the S346K/K347Y/K362Q, S346K1K347Y/K362H and S346I/K347D/K362H c-NADP-ME mutants did not show significant reductions in their K(m,NAD) values, the E314A mutation was then introduced into these triple mutants. Comparison of the kinetic parameters of each triple-quadruple mutant pair (for example, S346K/K347Y/K362Q versus E314A/S346K/K347Y/K362Q) revealed that all of the K(m) values for NAD(+) and NADP(+) of the quadruple mutants were significantly decreased, while either k(cat,NAD) or k(cat,NADP) was substantially increased. By adding the E314A mutation to these triple mutant enzymes, the E314A/S346K/K347Y/K362Q, E314A/S346K/K347Y/K362H and E314A/S3461/K347D/K362H c-NADP-ME variants are no longer debilitated but become mainly NAD utilizing enzymes by a considerable increase in catalysis using NAD(+) as the cofactor. These results suggest that abolishing the repulsive effect of Glu314 in these quadruple mutants increases the binding affinity of NAD(+) Here, we demonstrate that a series of E314A-containing c-NADP-ME quadruple mutants have been changed to NAD(+)-Utilizing enzymes by abrogating NADP(+) binding and increasing NAD(+) binding.
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