Structural and Kinetic Properties of NADP-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L. : PROTEINS, ENZYMES AND METABOLISM

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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 Km 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 NaHC0_3 on the activity of NADP-malic enzyme were small.
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