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 trinervia, to compare the pK values of its functional groups with those for the pigeon liver NADP-malic enzyme (Ml Schimerlik, WW Cleland [1977] Biochemistry 16: 576-583) and the plant NAD-malic enzyme (KO Willeford, RT Wedding ] 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 V,mx/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 V,,., remains constant. The log VmaxIKm 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 V,,, for the C4 enzyme. As in the case of the animal enzyme, the log Vm,x/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.Vma. up to pH 8.0 (7, 10), while the C3 plant enzyme appears to have an optimum activity around pH 6.4 to 7.3 (4, 14). The plant NAD-ME, located in mitochondria, is similar to the C3 NADP-ME with respect to the pH at which it exhibits maximum activity (15). Of course, several factors determine the optimum pH to use for in vitro assays, but a superficial look at all MEs suggests that the C4 enzyme is a special form that has evolved for decarboxylation at high rates in illuminated choroplasts during C4 photosynthesis.Extensive mechanistic information is available for the pigeon liver NADP-ME (7, 10) and is becoming available for plant NAD-ME (15), but such information for the forms of plant NADP-ME is minimal. In light of the differences in the pH optima for the various MEs, we have used the effect of pH on the kinetic parameters with respect to malate for the F. trinervia enzyme as a means of comparing the pK values of functional groups of this putative special form of ME with those published for the pigeon liver enzyme and the plant NAD-ME. MATERIALS AND METHODS Plant MaterialPlants of Flaveria trinervia (Spreng.) C. Mohr were grown in a greenhouse under a 14 h photoperiod during the summer months. The seed was collected locally, and the plants were fertilized with a Hoagland solution three times a week.

NADP‐dependent malate dehydrogenase (decarboxylating) from sugar cane leaves

“…Previous studies with NADP-dependent malate dehydrogenase (decarboxylating) from C4 plants showed that structural [7,8] and kinetic [8,12] properties ofthe protein are different depending on the conditions in the medium, with pH being an important effector of this. The results shown here on inhibition by NaCl and other salts suggest that ionic strength is also an important component in the determination of malate dehydrogenase activity.…”

Mitochondrial NAD+-dependent malic enzyme from Anopheles stephensi: a possible novel target for malaria mosquito control

“…These results suggest that the extent of inhibition was dependent on the size of the analogues (2 to 4-carbons), the presence of two carboxyl groups along with a 2-hydroxyl or 2-keto moiety important for binding of the substrate analogue to the enzyme. In a study performed with the enzyme from Flaveria trinervia , the presence of a group with a low p K value (6 to 6.6), probably an H residue, was responsible for the binding of the 2-hydroxyl of malate and transfer of the hydride to form the intermediate oxaloacetate [ 41 ]. These results suggest that the extent of inhibition was dependent on the size of the analogues (2 to 3-carbons), the presence of two carboxyl groups along with a 2-hydroxyl or 2-keto moiety important for binding of the substrate analogue to the enzyme.…”

Kinetic mechanism of NADP-malic enzyme from maize leaves

“…Since the pH optimum for the oxaloacetate decarboxylation and Lmalate oxidative decarboxylation reactions are different, the existence of an enzyme residue with pK 5.4-6.0 was proposed. Like the pigeon liver malic enzyme, the C4 NADP-ME has a group, probably a histidyl residue, with a pK of about 5.9-6.6 which binds to malate and facilitates subsequent catalysis (Holaday and Lowder 1989). Therefore, we can conclude that this group must be protonated for oxaloacetate decarboxylation (pH acid) and tmprotonated for malate oxidative decarboxylation (pH alkaline).…”