Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli. Purification to homogeneity and some properties

1980 ◽  
Vol 58 (1) ◽  
pp. 40-48 ◽  
Author(s):  
E. B. Waygood ◽  
T. Steeves
Keyword(s):  
Escherichia Coli ◽  
Protein Concentration ◽  
Phosphotransferase System ◽  
Temperature Dependent ◽  
Extra Copy ◽  
Ph Optimum ◽  
Low Protein ◽  
Kinetic Activity ◽  
Ping Pong ◽  
Enzyme I

Enzyme I of the phosphoenolpyruvate – sugar phosphotransferase system (PTS) has been purified to homogeneity from Escherichia coli. A merodiploid strain P650 which had an extra copy of the gene for enzyme I resulting in a twofold increase in the amount of activity was used. The enzyme is a dimer of 67 000 ± 5000 molecular weight subunits. At low protein concentration and 4 °C the monomer predominates, while at room temperature the dimer predominates. At higher protein concentrations (2 to 10 mg) this reversible temperature-dependent association-dissociation is not found. Enzyme I has a pH optimum of pH 7.2, a Km for HPr of 9 ± 3 μM, a Km for phosphoenolpyruvate of 0.18 ± 0.04 mM, and kinetics that are consistent with a bi bi Ping-Pong mechanism. No allosteric regulation of kinetic activity has been found. The amino acid composition has been determined and the ε1%280 nm is 4.4. Evidence suggests that the phosphorylated form of enzyme I is more stable.

Isozymes of α-galactosidase from Bacillus stearothermophilus

1980 ◽  
Vol 26 (8) ◽  
pp. 978-984 ◽  
Author(s):  
Dennis M. Pederson ◽  
Richard E. Goodman
Keyword(s):  
Gel Electrophoresis ◽  
Half Life ◽  
Protein Concentration ◽  
Mixed Type ◽  
Bacillus Stearothermophilus ◽  
Single Band ◽  
Molecular Forms ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Low Protein

Two molecular forms of α-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. α-Galactosidase I (with the substrate p-nitrophenyl α-D-galactopyranoside (PNPG)) has a pH optimum of 6 and a half-life at 65 °C of > 2 h at low protein concentration. α-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 °C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the β-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are α-galactosidase I, 280 000 ± 30 000 and α-galactosidase II, 325 000 ± 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 + 500 for α-galactosidase I and 84 000 ± 500 for α-galactosidase II, suggest that both enzymes consist of four subunits.

A novel phosphoprotein dependent on the bacterial phosphoenolpyruvate–sugar phosphotransferase system

1983 ◽  
Vol 61 (2-3) ◽  
pp. 150-153 ◽  
Author(s):  
E. Bruce Waygood ◽  
Roshan L. Mattoo
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Growth Conditions ◽  
Phosphotransferase System ◽  
Crude Extracts ◽  
Enzyme I ◽  
Novel Protein

A protein has been found by isoelectricfocusing and autoradiography in Escherichia coli and Salmonella typhimurium which was phosphorylated by enzyme I and an histidine-containing phosphocarrier protein (HPr) of the phosphoenolpyruvate–sugar phosphotransferase system (PTS). This protein was not factor IIIglc nor was it specifically induced by fructose. Its presence in soluble crude extracts was dependent upon growth conditions; however, the two bacteria had different patterns and amounts in respect to this novel protein. The protein was present in S. typhimurium SB2950 which has an extensive deletion through the pts operon, thus indicating that it must be coded for elsewhere on the genome.

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