The effect of pH on the covalent and metabolic control of C4 phosphoenolpyruvate carboxylase from Sorghum leaf.
The influence of pH on the in vitro activity and regulatory properties of Sorghum leaf C4 phosphoenolpyruvate carboxylase (PEPC) was investigated with respect to the phosphorylation status of the enzyme. In vitro protein phosphorylation was achieved using the catalytic subunit of a cAMP-dependent protein kinase (PKA) and a recombinant, immunopurified PEPC (0.9 mol of covalent Pi/mol PEPC subunit). Between pH 6.8 and 8, velocity and IC50 for L-malate increased for both the nonphosphorylated and the phosphorylated forms. With respect to the nonphosphorylated 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 for 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 PEPC caused a predominant V effect or a mixed (V/IC50) effect at pH 7.1 or 7.3, respectively. This was also observed with the enzyme from desalted crude protein extracts from dark or light-adapted Sorghum leaves and leaf-derived mesophyll protoplasts illuminated in the presence of methylamine, a compound known to increase cytosolic pH (pHc). At pH 7.3, desensitization to L-malate of phospho-PEPC was due to an enhanced ability of PEP to compete with the inhibitor. The positive effector glucose-6P acted similarly to phosphorylation; however, a combination of both factors (glucose-6P and phosphorylation) led to a much larger increase in the IC50 for L-malate than that observed by a single factor.(ABSTRACT TRUNCATED AT 250 WORDS)
Echevarria
C
C
Laboratorio de Fisiologia Vegetal, Universidad de Sevilla, Faculdad de Biologia, Spain.
Pacquit
V
V
Bakrim
N
N
Osuna
L
L
Delgado
B
B
Arrio-Dupont
M
M
Vidal
J
J
D005958:Glucosephosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008293:Malates / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D010788:Photosynthesis; D010944:Plants / Q000201:enzymology*
D005958:Glucosephosphates; D008293:Malates; D010730:Phosphoenolpyruvate Carboxylase