title: The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus.
abstract: Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.
mesh_terms: D002241:Carbohydrates / Q000494:pharmacology; D002845:Chromatography / Q000379:methods; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005941:Glucokinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D005976:Glutaral; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011489:Protein Denaturation; D013379:Substrate Specificity
chemical_list: D002241:Carbohydrates; D008670:Metals; D005941:Glucokinase; D005976:Glutaral