NADP-malic enzyme from the C-4 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 C-4 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 K-m for NAD is very high (20 mM), while the V-max is 50% greater than the V-max with NADP under the same conditions, The NAD dependent activity is competitively inhibited by micromolar concentrations of NADP and NADPH (K-i similar to 2 mu M). This very low K-i reflects the high affinity of malic enzyme for NADP(H) under these conditions. When utilizing NADP, the K-m for NADP is 1.5 mu M while the K-i for NADPH is 2 mu M. 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 K-m 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 K-m for Mn2+ is 4 mu M, whereas with NAD the K-m is 300 mu M. 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. (C) 1997 Academic Press.