Regulation of C3-enzymes in facultative phototrophic bacteria

Pyruvate kinase (EC2.7.1.40) from Rhodopseudomonas sphaeroides was purified 40-fold by precipitation with protamine sulfate and ammonium sulfate followed by gelfiltration. The preparations obtained from cells grown with different carbon sources or cultural conditions differ with respect to specific activity but not with respect to molecular weight (250000 dalton) or regulatory properties. The phosphoenolpyruvate (PEP)-saturation curve of the enzyme is sigmoidal with Hill coefficients varying from n _H =1.8 (pH 9.2) to 2.7 (pH 6.0). The enzyme is activated by adenosinemonophosphate (AMP) and the sugarmonophosphates ribose-5-phosphate (R-5-P), glucose-6-phosphate (G-6-P), and-to a lesser extent-fructose-6-phosphate (F-6-P). Fructose-1.6-bisphosphate (FDP) has no measurable effect. Inhibitors of the enzyme are adenosintriphosphate (ATP), inorganic phosphate (P _i ) and the dicarboxylic acids succinate and fumarate. Kinetic analysis reveals that the sugar-phosphates and the dicarboxylic acids act as true allosteric ligands, wheras the effects of AMP, ATP, and P _i cannot be interpreted solely in terms of allosteric interactions. Cold-treatment of the enzyme leads to a rapid loss of activity, but does not change the regulatory properties of the enzyme. Analysis of the kinetics of cold-inactivation and its reversal at 30°C, together with studies on the gelfiltration behaviour of the native and the cold-treated enzyme make it likely that the cold-induced loss of activity is due to a dissociation of the enzyme.