title: The inhibition of glucokinase and glycerokinase from Bacillus stearothermophilus by the triazine dye Procion Blue MX-3G.
abstract: Glucokinase from Bacillus stearothermophilus was irreversibly inactivated by the reactive dichlorotriazinyl dye Procion Blue MX-3G at pH 8.0. The enzyme was protected from inactivation by the substrate MgATP. Kinetic data implied that the dye occupied the MgATP-binding site. The apparent Km values for MgATP and D-glucose were found to be 70 microM and 210 microM respectively, and the Kd of the pure reactive dye was 16 microM; 1 mol of the pure reactive dye bound to 1 mol of glucokinase subunit. The dye was shown to have potential as an affinity probe for glucokinase. Glycerokinase from the same bacterium was inactivated by Procion Blue MX-3G at high concentrations (5 mM), but only after a period of increased enzyme activity. Kinetic data indicated that the dye preferentially attacked the glycerol-binding site. The apparent Km values for MgATP and glycerol were found to be 38 microM and 13 microM respectively, and 4 mol of reactive dye could be bound to 1 mol of glycerokinase subunit. This was surprising in view of the MgATP-dependent elution of glycerokinase from immobilized Procion Blue MX-3G.
authors.affiliation: Microbial Technology Laboratory, P.H.L.S. Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K.
authors.forename: C R
authors.initials: CR
authors.lastname: Goward
authors.forename: M D
authors.initials: MD
authors.lastname: Scawen
authors.forename: T
authors.initials: T
authors.lastname: Atkinson
mesh_terms: D001665:Binding Sites; D004396:Coloring Agents / Q000494:pharmacology*; D001411:Geobacillus stearothermophilus / Q000187:drug effects / Q000201:enzymology*; D005941:Glucokinase / Q000037:antagonists & inhibitors*; D005991:Glycerol Kinase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D010770:Phosphotransferases / Q000037:antagonists & inhibitors*
chemical_list: D004396:Coloring Agents; D010770:Phosphotransferases; D005941:Glucokinase; D005991:Glycerol Kinase