Abstract
The human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent malic enzyme has remarkable effects on the binding of NADP+ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The DeltaG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP+-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP+-specific.
Highlights
Study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP؉dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP؉-specific
Previous studies with other enzymes revealed that an Asp residue near the active site is expected to have an NADϩ preference and cannot tolerate the additional phosphate group of NADPϩ [36, 37]
The structures show that Asp345 is pointed away from the ribose and is ion-paired with Arg354 [10]. This provides an explanation for the observation that some malic enzymes with a conserved Asp at this position can still use NADPϩ as the cofactor
Summary
Study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP؉dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP؉-specific. Mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. Cooperative Effect of Malate on Wild-type and Mutant m-NAD-MEs—The initial velocities of m-NAD-ME measured in various concentrations of malate with either NADϩ or NADPϩ as the cofactor demonstrated sigmoidal kinetics (Fig. 2, closed circles), which implies cooperative malate binding.
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