Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The fructose-2,6-bisphosphatase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to be structurally and functionally homologous to phosphoglycerate mutase. Both enzymes catalyze their reactions via phosphoenzyme intermediates which utilize an active site histidine as a nucleophilic phosphoacceptor and another histidine as a proton donor to the leaving group. Glu327 in the bisphosphatase domain of the rat liver bifunctional enzyme is conserved in all phosphoglycerate mutase structures and is postulated, by modelling studies, to be located in the active site. Glu327 was mutated to Ala, Gln, or Asp. The mutant and wild-type enzymes were expressed in Escherichia coli with a T-7 RNA polymerase-based expression system and purified to homogeneity by substrate elution from phosphocellulose. The Glu327 mutants had apparent molecular weights of 110,000 by gel filtration and had unaltered 6-phosphofructo-2-kinase activity. Circular dichroism showed that the secondary structure of the Glu327 mutant enzyme forms was the same as the wild-type enzyme. The maximal velocity of the fructose-2,6-bisphosphatase of the Glu327----Ala, Glu327----Gln, and Glu327----Asp mutants was 4, 2, and 20%, respectively, that of the wild-type enzyme, but the rate of phosphoenzyme formation of the mutants was reduced by at least a factor of 1000. In addition, the rate constants of phosphoenzyme hydrolysis for the Glu372----Ala and Glu327----Gln mutants were 2.7 and 1.3%, respectively, of the wild type, whereas the rate constant for the Glu327----Asp mutant was 60% of the wild-type value. Glu327 was not a substrate or product binding site determinant since the Km for fructose-2,6-bisphosphate and Ki for fructose-6-phosphate of the mutants were not appreciably changed. The results implicate Glu327 as part of a catalytic triad in fructose-2,6-bisphosphatase and suggest that it influences the protonation state of the active site histidine residues during phosphoenzyme formation and/or acts as a base catalyst to enhance the nucleophilic attack of water on the phosphoenzyme intermediate.
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PMID:Glu327 is part of a catalytic triad in rat liver fructose-2,6-bisphosphatase. 131 12

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.
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PMID:Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase. 142 31

A number of mammalian enzymes have been expressed in Escherichia coli using the T7 RNA polymerase system, but the production of large amounts of these proteins has been limited by the low percentage of active enzyme that is found in the soluble fraction. In this report the effect of induction temperature was tested on the recovery of four rat liver enzymes, 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, fructose-2,6-bisphosphatase, glucokinase, and fructose-1,6-bisphosphatase. We also tested the effect using a host cell strain that contains a plasmid encoding T7 lysozyme, an inhibitor of T7 RNA polymerase. Large amounts of the first three enzymes accumulated in the cells after 4 h of induction at 37 degrees C, but only about 1-2% of the total expressed proteins were recovered in a soluble, active form. When the induction was carried out at 22 degrees C for 48 h with the pLysS strain, 20- to 30-fold higher amounts of the active expressed enzymes were recovered in the soluble fraction, even though the total accumulation and the rate of synthesis of these proteins were reduced. The optimal concentration of isopropyl-1-thio-beta-D-galactopyranoside required for induction was the same at both temperatures. On the other hand, the recovery of active fructose-1,6-bisphosphatase, a heat-stable enzyme, was 66% at 37 degrees C and was essentially unchanged at an induction temperature of 22 degrees C. Lowered induction temperature would appear to be of utility for enhanced recovery of active mammalian enzymes which are insoluble in E. coli cytosol at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of mammalian liver glycolytic/gluconeogenic enzymes in Escherichia coli: recovery of active enzyme is strain and temperature dependent. 196 24

The rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (ATP:D-fructose-6-phosphate 2-phosphotransferase/D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 2.7.1.105/EC 3.1.3.46) and its separate kinase domain were expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The bifunctional enzyme (470 residues per subunit) was efficiently expressed as a protein that starts with the initiator methionine residue and ends at the carboxyl-terminal tyrosine residue. The expressed protein was purified to homogeneity by anion exchange and Blue Sepharose chromatography and had kinetic and physical properties similar to the purified rat liver enzyme, including its behavior as a dimer during gel filtration, activation of the kinase by phosphate and inhibition by alpha-glycerol phosphate, and mediation of the bisphosphatase reaction by a phosphoenzyme intermediate. The expressed 6-phosphofructo-2-kinase also started with the initiator methionine but ended at residue 257. The partially purified kinase domain was catalytically active, had reduced affinities for ATP and fructose 6-phosphate compared with the kinase of the bifunctional enzyme, and had no fructose-2,6-bisphosphatase activity. The kinase domain also behaved as an oligomeric protein during gel filtration. The expression of an active kinase domain and the previous demonstration of an actively expressed bisphosphatase domain provide strong support for the hypothesis that the hepatic enzyme consists of two independent catalytic domains encoded by a fused gene.
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PMID:Expression of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its kinase domain in Escherichia coli. 255 38

The fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (EC 2.7.105/EC 3.1.3.46) was expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The protein was efficiently expressed (i) as a fusion protein that starts at the T7 major capsid protein initiation site in a pET expression vector and (ii) as a protein that starts within the bisphosphatase sequence by translation reinitiation. Both proteins have similar properties. The protein was purified to homogeneity by anion-exchange chromatography and gel filtration. The purified fructose-2,6-bisphosphatase domain was active and no 6-phosphofructo-2-kinase activity was found associated with it. In contrast to the dimeric bifunctional enzyme, the fructose-2,6-bisphosphatase domain behaved as a monomer of 30 kDa. The turnover number and kinetic properties of the separate bisphosphatase domain were similar to those of the bisphosphatase of the bifunctional enzyme, including the ability to form a phosphoenzyme intermediate. These results support the hypothesis that the rat liver enzyme consists of two independent domains and is a member of a class of enzymes formed by gene fusion.
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PMID:Expression of the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli. 284 83

Chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was expressed in E. coli by using a pET3a T7 RNA polymerase-based expression system and was purified to homogeneity. The kinase and bisphosphatase of the expressed bifunctional enzyme had kinetic properties identical to those of the native chicken liver enzyme. However, the kinase activity of the chicken liver enzyme was 7-fold higher, while the bisphosphatase activity was 50 percent lower than those of the rat liver enzyme. Cys-256 of the rat liver bisphosphatase domain is not conserved in the chicken liver enzyme. A site-directed mutation was engineered at Cys-256 of the rat liver enzyme and the results indicate that the variation of this residue is not responsible for the difference in fructose-2,6-bisphosphatase activity between the rat and chicken liver enzymes. It is postulated that the difference in the kinase/bisphosphatase activity ratios of these two enzymes results from differences in their NH2-terminal regions.
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PMID:Expression of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli. 773 80

The nucleotide sequence of 1981 bp cDNA containing the entire coding region of a human placental fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase was determined. The sequence encodes 469 amino acids and, based on homology to the rat testis enzyme, appears to be the testis-type isozyme expressed in placenta. The enzyme was expressed in Escherichia coli BL21 (DE3) by using a T7 RNA polymerase-based expression system and purified to homogeneity. The expressed enzyme was bifunctional with specific activities of 75 and 80 mU/mg of kinase and phosphatase, respectively. Kinetic parameters of the expressed enzyme are similar to those of the rat testis enzyme.
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PMID:Human placental fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase: its isozymic form, expression and characterization. 940 80