Effect of pH on the Kinetic Parameters of NADP-Malic Enzyme from a C4Flaveria (Asteraceae) Species

Abstract

We have used the pH variation in the kinetic parameters with respect to malate of NADP-malic enzyme purified from the C4 species, Flaveria trinerval, 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 C4 enzyme has a group with a pK of about 6.0 (6.6 for the C4 enzyme), as indicated from plots of the log Vmax/Km (Vmax = maximum rate of catalysis) versus pH, which must lose a proton for malate binding and subsequent catalysis. The optimum ionization for the C4 enzyme-NADP-Mg2+ complex occurs at pH 7.1 to 7.5. From pH 7.5 to 8.4, the Km increases, but Vmax remains constant. The log Vmax/ Km 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 Km and a decrease in Km and a decrease in Vmax for the C4 enzyme. As in the case of the animal enzyme, the log Vmax/Km plot for the C4 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 C4 NADP-malic enzyme to maintain high activity at pH 8.0 in illuminated chloroplasts.