NADP–Malic Enzyme from the C4PlantFlaveria bidentis:Nucleotide Substrate Specificity

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NADP–malic enzyme (NADP–ME, EC 1.1.1.40) was purified to near-homogeneity from leaves of the C4dicotFlaveria bidentisand 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+. TheKmfor NAD is very high (20 mm), while theVmaxis 50% greater than theVmaxwith NADP under the same conditions. The NAD-dependent activity is competitively inhibited by micromolar concentrations of NADP and NADPH (Ki∼2 μm). This very lowKireflects the high affinity of malic enzyme for NADP(H) under these conditions. When utilizing NADP, theKmfor NADP is 1.5 μmwhile theKifor NADPH is 2 μm. 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 theKmvalues 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 substratel-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 asl-malate. With NADP as substrate theKmfor Mn2+is 4 μm, whereas with NAD theKmis 300 μm. 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.