Regulation of C3-enzymes in facultative phototrophic bacteria: the cold-labile pyruvate kinase of Rhodopseudomonas sphaeroides.
Pyruvate kinase (EC2.7.1.40) from Rhodopseudomonas sphaeroides was purified 40-fold be precipitation with protamine sulfate and ammonium sulfate followed by gel-filtration. 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 cruve of the enzyme is sigmoidal with Hill coefficients varying from nH equals 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 adenosinetriphosphate (ATP), inorganic phosphate (Pi) and the dicarboxylic acids succinate and fumarate. Kinetic analysis reveals that the sugar-phosphates and the dicarboxylic acids act as true allosteric ligands, whereas the effects of AMP, ATP, and Pi cannot be interpreted soley in terms of allosteric interactions. Cold-treatment of the enzymes lead 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 percent 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.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D002413:Cations, Divalent; D002474:Cell-Free System; D002850:Chromatography, Gel; D003080:Cold Temperature; D003470:Culture Media; D004355:Drug Stability; D004789:Enzyme Activation; D005650:Fumarates / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D010710:Phosphates / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011189:Potassium Chloride; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013386:Succinates / Q000494:pharmacology; D013403:Sugar Phosphates / Q000494:pharmacology
D002413:Cations, Divalent; D003470:Culture Media; D005650:Fumarates; D008024:Ligands; D010710:Phosphates; D013386:Succinates; D013403:Sugar Phosphates; D000249:Adenosine Monophosphate; D011189:Potassium Chloride; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D005947:Glucose