Kinetic analysis of the non-phosphorylated, in vitro phosphorylated, and phosphorylation-site-mutant (Asp8) forms of intact recombinant C4 phosphoenolpyruvate carboxylase from sorghum

Autor: Stephen M. G. Duff, Carlos S. Andreo, Valerie Pacquit, Loic Lepiniec, Gautam Sarath, Shirley A. Condon, Jean Vidal, Pierre Gadal, Raymond Chollet
Přispěvatelé: Unité associée au Département Biologie et Amélioration des Plantes (Associée BAP), Institut National de la Recherche Agronomique (INRA), ProdInra, Migration
Rok vydání: 1995
Předmět:
Zdroj: European Journal of Biochemistry
European Journal of Biochemistry, Wiley, 1995, 228, pp.92-95
ISSN: 0014-2956
1432-1327
Popis: Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8--Asp) forms of purified recombinant sorghum C4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.31) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent Ki for the negative allosteric effector L-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent Ka for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1 mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and Km (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by L-malate and, most especially, Ka (Glc6P) and Ki (L-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C4-photosynthesis enzyme.
Databáze: OpenAIRE
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