Effect of pH on the Kinetic Parameters of NADP-Malic Enzyme from a C(4)Flaveria (Asteraceae) Species.

We have used the pH variation in the kinetic parameters with respect to malate of NADP-malic enzyme purified from the C(4) species, Flaveria trinervia, to compare the pK values of its functional groups with those for the pigeon liver NADP-malic enzyme (MI Schimerlik, WW Cleland [1977] Biochemistry 16: 576-583) and the plant NAD-malic enzyme (KO Willeford, RT Wedding [1987] Plant Physiol 84: 1084-1087). Like the other enzymes, the C(4) enzyme has a group with a pK of about 6.0 (6.6 for the C(4) enzyme), as indicated from plots of the log V(max)/K(m) (V(max) = maximum rate of catalysis) versus pH, which must lose a proton for malate binding and subsequent catalysis. The optimum ionization for the C(4) enzyme-NADP-Mg(2+) complex occurs at pH 7.1 to 7.5. From pH 7.5 to 8.4, the K(m) increases, but V(max) remains constant. The log V(max)/K(m) plot in this pH range indicates a group with a pK of about 7.7. The other malic enzymes exhibit a similar pK. Above pH 8.4, deprotonation leads to a marked increase in K(m) and a decrease in V(max) for the C(4) enzyme. As in the case of the animal enzyme, the log V(max)/K(m) plot for the C(4) enzyme appears to approach a slope of two. The curve suggests an average pK of 8.4 for the groups involved, while the animal enzyme exhibits an average pK of 9.0. The NAD-malic enzyme does not exhibit any pK values at these high pK values. We hypothesize that the putative groups with the high pK values may be at least partially responsible for the ability of the C(4) NADP-malic enzyme to maintain high activity at pH 8.0 in illuminated chloroplasts.