Structural and kinetic properties of NADP-malic enzyme from the inducible crassulacean acid metabolism plant mesembryanthemum crystallinum l.

NADP-malic enzyme (EC 1.1.1.40), which is involved in Crassulacean acid metabolism (CAM), was purified to electrophoretic homogeneity from the leaves of the inducible CAM plant Mesembryanthemum crystallinum. The NADP-malic enzyme, which was purified 1,146-fold, has a specific activity of 68.8 μmol (mg protein)^-1 min^-1. The molecular weight of the subunits of the enzyme was 64 kDa. The native molecular weight of the enzyme was determined by gel-filtration to be 390 kDa, indicating that the purified NADP-malic enzyme is a hexamer of identical subunits. The optimal pH for activity of the enzyme was around 7.2. Double-reciprocal plots of the enzymatic activity as a function of the concentration of L-malate yielded straight lines both at pH 7.2 and at pH 7.8 and did not reveal any evidence for cooperativity of binding of L-malate. The K_m value for L-malate was 0.35 mM. Hill plots of the activity as a function of the concentration of NADP⁺ indicated positive cooperativity in the binding of NADP⁺ to the enzyme with a Hill coefficient (nH) of 2.0. An S_0.5 value (the concentration giving half-maximal activity) of 9.9 μM for NADP⁺ was obtained. Oxaloacetate inhibited the activity of the NADP-malic enzyme. Effects of succinate and NaHCO₃ on the activity of NADP-malic enzyme were small.