Kinetic Analysis of the Non-Phosphorylated, In Vitro Phosphorylated, and Phosphorylation-Site-Mutant (Asp8) Forms of Intact Recombinant C₄ Phosphoenolpyruvate Carboxylase From Sorghum

Authors

Stephen M. G. Duff, University of Nebraska–Lincoln
Carlos S. Andreo, University of Nebraska–Lincoln
Valérie Pacquit, Institut de Biotechnologie des Plantes, Université de Paris-Sud, Orsay, France Centro de Estudios
Loïc Lepiniec, Institut de Biotechnologie des Plantes, Université de Paris-Sud, Orsay, France Centro de Estudios
Gautam Sarath, University of Nebraska–LincolnFollow
Shirley A. Condon, Institut de Biotechnologie des Plantes, Université de Paris-Sud, Orsay, France Centro de Estudios
Jean Vidal, Institut de Biotechnologie des Plantes, Université de Paris-Sud, Orsay, France Centro de Estudios
Pierre Gadal, University of Nebraska–Lincoln
Raymond Chollet, University of Nebraska–LincolnFollow

ORCID IDs

Gautam Sarath

Date of this Version

1995

Comments

Published in European Journal of Biochemistry 228 (1995), pp. 92–95. Copyright © 1995 FEBS. Used by permission.

Abstract

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 4.1.1.3 1) 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 K_i/ for the negative allosteric effector l-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent K_a for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1mM 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 K_m (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by l-malate and, most especially, K_a (Glc6P) and K_iv (l-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C₄-photosynthesis enzyme.