NADP-malic enzyme from the C4 plant Flaveria bidentis: nucleotide substrate specificity.
NADP-malic enzyme (NADP-ME, EC 1.1.1.40) was purified to near-homogeneity from leaves of the C4 dicot Flaveria bidentis and shown to possess intrinsic NAD-dependent malic enzyme activity. The NAD-dependent activity is optimal at pH 7.5 and in the presence of Mn2+. The Km for NAD is very high (20 mM), while the Vmax is 50% greater than the Vmax with NADP under the same conditions. The NAD-dependent activity is competitively inhibited by micromolar concentrations of NADP and NADPH (Ki approximately 2 microM). This very low Ki reflects the high affinity of malic enzyme for NADP(H) under these conditions. When utilizing NADP, the Km for NADP is 1.5 microM while the Ki for NADPH is 2 microM. Chicken liver NADP-ME also has NAD-dependent activity that is inhibited by low concentrations of NADPH. These results indicate that the NAD- and NADP-dependent activities are likely catalyzed by the same active site. The use of NAD as an alternative coenzyme revealed interactions between the binding of coenzyme and metal ions on the Km values of each of the other participants in the malic enzyme reaction. Thus, the affinity of malic enzyme for the divalent metal ions Mg2+ and particularly Mn2+ as well as the other substrate L-malate is also dependent on the nucleotide coenzyme substrate. In turn, the divalent metal ion influences the affinity of the enzyme for the coenzyme as well as L-malate. With NADP as substrate the Km for Mn2+ is 4 microM, whereas with NAD the Km is 300 microM. The relatively high affinity of the enzyme for Mn2+ and low affinity for NAD required the use of metal ion buffers when determining these values because of the substantial depletion of free Mn2+ caused by binding to NADP.
Ashton
A R
AR
CSIRO Plant Industry, Canberra, Australia. tony.ashton@pi.csiro.au
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002645:Chickens; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010788:Photosynthesis; D010944:Plants / Q000201:enzymology*; D013045:Species Specificity; D013379:Substrate Specificity
D002413:Cations, Divalent; D008293:Malates; D009243:NAD; D009249:NADP; C030298:malic acid; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+); C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)