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Query: EC:4.1.1.32 (phosphoenolpyruvate carboxykinase)
 4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Purification and characterization of recombinant phosphoenolpyruvate carboxylase of Thermus sp. 890 15

A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, by screening a genomic DNA library using the coding region of Anacystis nidulans 6301 PEPC as a probe. The S. vulcanus PEPC gene (SvPEPC) had an open reading frame for a polypeptide of 1,011 amino acid residues with a calculated molecular mass of 116.4 kDa. SvPEPC was expressed in E. coli BL21 Codonplus (DE3), using pET32a as a vector. The purified recombinant SvPEPC protein with a tag showed a single band of 120 kDa on SDS-PAGE. The enzyme forms homotetramer as judged by gel filtration. SvPEPC retained full activity even after incubation at 50 degrees C for 60 min or exposure to 0.5 M guanidine-HCl at 30 degrees C for 20 h, being more stable than C4-form PEPC from Zea mays (ZmPEPC(C4)). SvPEPC activity showed a sharp optimum temperature of 42 degrees C at pH 7.5 and an optimum pH of 9.0 at 30 degrees C. The enzyme, unlike most plant PEPCs, was predominantly activated by fructose 1,6-bisphosphate (Fruc-1,6-P(2)), and slightly stimulated by 3-phosphoglycerate (3-PGA), glucose 6-phosphate (Gluc-6-P), glucose 1-phosphate, Glu and Gln. Acetyl-CoA known as a strong activator of most bacterial PEPCs but not of plant PEPCs, showed no effect on the enzyme activity. SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs. I(0.5) for Asp was increased about 2-fold by Gluc-6-P while markedly decreased by Fruc-1,6-P(2), Glu and Gln about 3- to 4-fold. The regulation mechanism of SvPEPC is not readily interpretable by conventional allosteric models.
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PMID:Molecular characterization of a phosphoenolpyruvate carboxylase from a thermophilic cyanobacterium, Synechococcus vulcanus with unusual allosteric properties. 1186 95