THE EFFECT OF PH ON THE COVALENT AND METABOLIC CONTROL OF C-4 PHOSPHOENOLPYRUVATE CARBOXYLASE FROM SORGHUM LEAF

The influence of pH on the in vitro activity and regulatory properties of Sorghum leaf C-4 phosphoenolpyruvate carboxylase (PEPC) was invest igated with respect to the phosphorylation status of the enzyme. In vi tro protein phosphorylation was achieved using the catalytic subunit o f a cAMP-dependent protein kinase (PKA) and a recombinant, immunopurif ied PEPC (0.9 mol of covalent P-i/mol PEPC subunit). Between pH 6.8 an d 8, velocity and IC50 for L-malate increased for both the nonphosphor ylated and the phosphorylated forms. With respect to the nonphosphoryl ated PEPC, the phospho-PEPC always gave high values for these kinetic parameters at the pH range investigated, especially between pH 7 and 7 .3. The phosphorylation-induced stimulation of PEPC activity was four- to fivefold at pH 7.1 and approximately twofold at pH 7.3. The IC50 f or L-malate showed a two- to threefold increase at pH 7.3, but varied less at pH 7.1 upon PEPC phosphorylation. Thus, phosphorylation of PEP C caused a predominant V effect or a mixed (V/IC50) effect at pH 7.1 o r 7.3, respectively. This was also observed with the enzyme from desal ted crude protein extracts from dark or light-adapted Sorghum leaves a nd leaf-derived mesophyll protoplasts illuminated in the presence of m ethylamine, a compound known to increase cytosolic pH (pHc). At pH 7.3 , desensitization to L-malate pf phospho-PEPC was due to an enhanced a bility of PEP to compete with the inhibitor. The positive effector glu cose-6P acted similarly to phosphorylation; however a combination of b oth factors (glucose-6P and phosphorylation) led to a much larger incr ease in the IC50 for L-malate than that observed by a single factor. T hese effects may account for the actual CO2 assimilation rate of an il luminated Sorghum leaf. In reconstituted assays performed at pH 7.1 or 7.3, in the presence of PKA, the addition of 10 mM L-malate decreased the phosphorylation rate of the PEPC. This effect was strongly inhibi ted by the addition of glucose-6P (5 mM). It is suggested that modulat ion of the phosphorylation rate is partly through conformational chang es in the PEPC induced by these photosynthetic metabolites. Together w ith previous findings indicating that the photoinduction of PEPC prote in kinase was strictly dependent on cytosolic alkalization in meosphyl l cell protoplasts (J. N. Pierre et al., 1992, Eur. J. Biochem. 210, 5 31-537), the present data support the view that cytosolic pH is an imp ortant component of the overall regulation of PEPC in C-4-photosynthes is. Finally, the role of phosphorylation appears to be a mechanism to amplify the regulatory influences of pHc and glucose-6P on PEPC activi ty. (C) 1994 Academic Press, Inc.