Goward C R, Scawen M D, Atkinson T
Microbial Technology Laboratory, P.H.L.S. Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K.
Biochem J. 1987 Aug 15;246(1):83-8. doi: 10.1042/bj2460083.
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.
嗜热脂肪芽孢杆菌的葡萄糖激酶在pH 8.0时被活性二氯三嗪基染料普施安蓝MX - 3G不可逆地失活。底物MgATP可保护该酶不被失活。动力学数据表明该染料占据了MgATP结合位点。发现MgATP和D - 葡萄糖的表观Km值分别为70 μM和210 μM，纯活性染料的Kd为16 μM；1摩尔纯活性染料与1摩尔葡萄糖激酶亚基结合。结果表明该染料有潜力作为葡萄糖激酶的亲和探针。来自同一细菌的甘油激酶在高浓度（5 mM）的普施安蓝MX - 3G作用下被失活，但只是在酶活性增加一段时间之后。动力学数据表明该染料优先攻击甘油结合位点。发现MgATP和甘油的表观Km值分别为38 μM和13 μM，4摩尔活性染料可与1摩尔甘油激酶亚基结合。鉴于甘油激酶从固定化的普施安蓝MX - 3G上的MgATP依赖性洗脱，这一结果令人惊讶。
