The Enzyme Kinetics of the NADP-Malic Enzyme from Tobacco Leaves

Abstract

Malic enzyme (L-malate: NADP + oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40, NADP-ME), which was found in chloroplasts, was isolated from tobacco leaves ( Nicotiana tabacum L.) almost homogenous. The specific enzyme activity was 0.95 μmol min -1 mg -1 . The enzyme pH optimum was found between pH 7.1 and 7.4. The affinity of NADP-ME to substrates (L-malate and NADP + ) was evaluated in the presence of divalent metal ions (Mg 2+ , Mn 2+ , Co 2+ , Ni 2+ ). The value of the apparent Michaelis constant of NADP-ME for L-malate was dependent on the ion cofactor, while no such relationship was found for NADP + . The dependence of the reaction rate on concentration of Mg 2+ indicates the presence of more than one binding site for these ions in NADP-ME. Likewise, the sigmoidal dependence of the reaction rate on Mn 2+ concentration and the value of Hill coefficient 7.5 indicate the positive cooperativity of the reaction kinetics in the presence of the ions. The effect of Co 2+ and Ni 2+ ions was analogous to that of Mn 2+ ions; however, the cooperativity was lower (the values of Hill coefficients were 3.0 and 1.3 for Co 2+ and Ni 2+ , respectively). Regulation of NADP-ME from tobacco leaves by divalent metal ions is discussed.