caa a22 4500 397556411 CHVBK 20180308164803.0 cr unu---uuuuu 161202e199609 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021444 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021444 Purification and Characterization of Recombinant Phosphoenolpyruvate Carboxylase of Thermus sp [Elektronische Daten] [Tsutomu Nakamura, Shigeru Minoguchi, Katsura Izui] 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, Therm us 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°C. The half-saturation concentrations for both substrates at 70°C were about twice those at 30°C. Half-lives of the enzyme at 85,90, and 95°C were 220, 110, and 50 mm, respectively. Thermus PEPC was highly tolerant also to guanidine hydrochloride (Gdn-HC1): the concentrations required for complete inactivation of Thermus and E. coli PEPCs after incubation at 30°C for 20 h were 3.5 and 0.8 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. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence allosteric effectors nationallicence guanidine hydrochloride nationallicence phosphoenolpyruvate carboxylase nationallicence thermostable enzyme nationallicence Thermus sp nationallicence Nakamura Tsutomu Department of Chemistry, Faculty of Science Sakyo-ku, Kyoto 606-01 aut Minoguchi Shigeru Department of Chemistry, Faculty of Science Sakyo-ku, Kyoto 606-01 aut Izui Katsura Department of Agricultural Biology, Faculty of Agriculture, Kyoto University Sakyo-ku, Kyoto 606-01 aut The Journal of Biochemistry Oxford University Press 120/3(1996-09), 518-524 0021-924X 120:3<518 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021444 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021444 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nakamura Tsutomu Department of Chemistry, Faculty of Science Sakyo-ku, Kyoto 606-01 aut NATIONALLICENCE 700 1- Minoguchi Shigeru Department of Chemistry, Faculty of Science Sakyo-ku, Kyoto 606-01 aut NATIONALLICENCE 700 1- Izui Katsura Department of Agricultural Biology, Faculty of Agriculture, Kyoto University Sakyo-ku, Kyoto 606-01 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/3(1996-09), 518-524 0021-924X 120:3<518 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford