Abstract
Malic enzyme (EC 1.1.1.40, NADP-ME) was isolated from tobacco leaves (Nicotiana tabacum L.) almost homogenous. The specific enzyme activity was 0.95 mol.min-1mg-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 (Mg2+, Mn2+, Co2+, Ni2+).
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 Mg2+ indicates the presence of more than one binding site for these ions in NADP-ME.
Likewise, the sigmoidal dependence of the reaction rate on Mn2+ 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 Co2+ and Ni2+ ions was analogous to that of Mn2+ ions; however, the cooperativity was lower.