Determinants of nucleotide-binding selectivity of malic enzyme

Abstract

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 S346I/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, S346K/K347Y/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/S346I/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. © 2011 Hsieh et al.