The kinetic mechanism of AAC(3)-IV aminoglycoside acetyltransferase from Escherichia coli
The kinetic mechanism of AAC(3)-IV aminoglycoside acetyltransferase from Escherichia coli
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DOI：
10.1021/bi051777d
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发表时间：
2005-12-13
期刊：
影响因子：
2.9
通讯作者：
Blanchard, JS
中科院分区：
文献类型：
--
作者：
Magalhaes, MLB;Blanchard, JS
来自大肠杆菌的氨基糖苷类 3-N-乙酰基转移酶 AAC(3)-IV 表现出非常广泛的氨基糖苷类特异性，导致对大量氨基糖苷类药物产生耐药性，包括非典型兽用抗生素安普霉素。我们在此报告了 AAC(3)-IV 催化的乙酰基转移反应的底物特异性和动力学机制的表征。稳态动力学参数揭示了酰基供体的狭窄特异性和氨基糖苷类的广泛活性。在迄今为止研究的所有 AAC(3) 中，AAC(3)-IV 具有最广泛的底物特异性。死端抑制和 ITC 实验表明 AAC(3)-IV 遵循顺序、随机双双动力学机制。对动力学参数的 pH 依赖性的分析揭示了酸和碱辅助催化以及参与底物结合的另外三个可电离基团的存在。溶剂动力学同位素效应的大小表明化学步骤在整个反应中至少部分地限制速率。
The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides, including the atypical veterinary antibiotic, apramycin. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV. The steady-state kinetic parameters revealed a narrow specificity for the acyl-donor and broad range of activity for aminoglycosides. AAC(3)-IV has the broadest substrate specificity of all AAC(3)'s studied to date. Dead-end inhibition and ITC experiments revealed that AAC(3)-IV follows a sequential, random bi-bi kinetic mechanism. The analysis of the pH dependence of the kinetic parameters revealed acid- and base-assisted catalysis and the existence of three additional ionizable groups involved in substrate binding. The magnitude of the solvent kinetic isotope effects suggests that a chemical step is at least partially rate limiting in the overall reaction.