Purification and characterization of recombinant phosphoenolpyruvate carboxylase of Thermus sp.
Recombinant phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) of an extreme thermophile, Thermus sp., which was expressed in Escherichia coli cells, was purified and its enzymological properties were investigated and compared with native Thermus PEPC. The enzyme activity was strongly dependent on acetyl-CoA, an allosteric activator, and inhibited by malate or aspartate. Contrary to the other known PEPCs, Thermus PEPC was not activated but rather inhibited by phosphorylated compounds such as fructose 1,6-bisphosphate and GTP. The specific activity in the presence of 0.3 mM acetyl-CoA and 2 mM phosphoenolpyruvate was highest at 70 degrees C. The half-saturation concentrations for both substrates at 70 degrees C were about twice those at 30 degrees C. Half-lives of the enzyme at 85, 90, and 95 degrees C were 220, 110, and 50 min, respectively. Thermus PEPC was highly tolerant also to guanidine hydrochloride (Gdn-HCl): the concentrations required for complete inactivation of Thermus and E. coli PEPCs after incubation at 30 degrees C for 20 h were 3.5 and 0.6 M, respectively. The properties of recombinant and native enzyme were similar to each other except for the catalytic activity after incubation with 1-2 M Gdn-HCl.
Department of Chemistry, Faculty of Science, Kyoto University.
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Nakamura
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Minoguchi
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Izui
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000494:Allosteric Regulation; D001224:Aspartic Acid / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008293:Malates / Q000494:pharmacology; D008970:Molecular Weight; D010730:Phosphoenolpyruvate Carboxylase / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D013824:Thermus / Q000201:enzymology*
D006146:Guanidines; D008293:Malates; D011994:Recombinant Proteins; D001224:Aspartic Acid; D000105:Acetyl Coenzyme A; C030298:malic acid; D010730:Phosphoenolpyruvate Carboxylase; D019791:Guanidine