McKay G A, Wright G D
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Biochemistry. 1996 Jul 2;35(26):8680-5. doi: 10.1021/bi9603884.
Bacterial resistance to the aminoglycoside antibiotics is manifested primarily through the production of enzymes which covalently modify these drugs. The Enterococci and Staphylococci produce an ATP-dependent kinase, APH(3')-IIIa, which phosphorylates such antibiotics as kanamycin, amikacin, and neomycin, and this enzyme shows a Theorell-Chance kinetic mechanism by traditional product and analogue inhibitor analysis and by the alternative substrate diagnostic [McKay, G. A., & Wright, G. D. (1995) J. Biol. Chem. 270, 24686-24692]. We report that the APH(3')-IIIa exhibits small solvent (VH/VD approximately equal to 1.50) and thio effects (VATP/VATP gamma S = 2) indicating hydroxyl group deprotonation and nucleophilic attack on ATP do not significantly contribute to the overall steady-state rate. The enzymatic rates were determined with the viscogens PEG 8000, glycerol, and sucrose, and these experiments demonstrate that ATP binding and ADP release are diffusion controlled and that ADP release is solely rate limiting for APH(3')-IIIa. In addition, the slope of V/K for ATP vs relative viscosity is greater than the theoretical limit of 1, suggesting a possible enzyme conformational change upon binding of ATP. This new experimental evidence supports a Theorell-Chance mechanism for APH(3')-IIIa.
细菌对氨基糖苷类抗生素的耐药性主要通过产生共价修饰这些药物的酶来体现。肠球菌和葡萄球菌产生一种ATP依赖性激酶APH(3')-IIIa，它可使卡那霉素、阿米卡星和新霉素等抗生素磷酸化，并且通过传统的产物和类似物抑制剂分析以及替代底物诊断，这种酶表现出Theorell-Chance动力学机制[麦凯，G.A.，& 赖特，G.D.（1995年）《生物化学杂志》270，24686 - 24692]。我们报告APH(3')-IIIa表现出较小的溶剂效应（VH/VD约等于1.50）和硫效应（VATP/VATPγS = 2），表明羟基去质子化和对ATP的亲核攻击对整体稳态速率的贡献不大。用粘性剂聚乙二醇8000、甘油和蔗糖测定酶促反应速率，这些实验表明ATP结合和ADP释放受扩散控制，并且ADP释放是APH(3')-IIIa唯一的限速步骤。此外，ATP的V/K对相对粘度的斜率大于理论极限1，这表明在ATP结合时可能发生酶构象变化。这一新的实验证据支持了APH(3')-IIIa的Theorell-Chance机制。
