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首页> 外文期刊>Biochemistry >PURIFICATION, CHARACTERIZATION, AND INVESTIGATION OF THE MECHANISM OF AMINOGLYCOSIDE 3'-PHOSPHOTRANSFERASE TYPE IA
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PURIFICATION, CHARACTERIZATION, AND INVESTIGATION OF THE MECHANISM OF AMINOGLYCOSIDE 3'-PHOSPHOTRANSFERASE TYPE IA

机译：IA型氨基糖苷3'-磷酸转移酶的纯化，鉴定和机理研究

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摘要

Aminoglycoside 3'-phosphotransferases [APH(3')s] are the most common cause of bacterial high-level resistance to aminoglycoside antibiotics in clinical isolates. A one-step affinity chromatography was used to purify APH(3') type Ia. The kinetic parameters for turnover of seven aminoglycosides and the corresponding minimum inhibitory concentrations for a strain of Escherichia coil harboring APH(3')-Ia were determined. The enzyme phosphorylates its substrates with k(cat)/K-m values of 10(6)-10(8) M(-1) S-1, including substrates such as amikacin and butirosin A which traditionally have been considered poor s substrates for this enzyme. The optimal pH for the phosphotransferase activity was observed to be 7.0-7.5. The purified enzyme was found to be prone to dimerization in the absence of a reducing agent. Treatment of the enzyme with trypsin excised a 4 kDa fragment from the N-terminus which contained the amino acid residue Cys-10. The 27 kDa proteolyzed APH(3')-Ia did not dimerize, suggesting that Cys-10 was involved in dimerization via a disulfide bond. The phophorylated kanamycin A was isolated, and the phosphorylation was confirmed to occur at the 3'-hydroxyl. Furthermore, both APH(3')-Ia and APH(3')-IIa were shown to phosphorylate water (''ATP hydrolase'' activity) at a rate of ca. 10(4)-10(6)-fold slower (effect on k(cat)/K-m) than that for the phosphoryl transfer to a typical aminoglycoside. The results of product-inhibition and alternative substrate diagnostics indicate an equilibrium-random mechanism for phosphorylation of aminoglycosides by APH(3')-Ia.
机译：氨基糖苷3'-磷酸转移酶[APH（3'）s]是临床分离物中细菌对氨基糖苷抗生素产生高水平耐药性的最常见原因。一步亲和色谱用于纯化Ia型APH（3'）。确定了动力学参数的七个氨基糖苷的营业额和相应的最小抑制浓度为带有APH（3'）-Ia的大肠杆菌的菌株。该酶以k（cat）/ Km值为10（6）-10（8）M（-1）S-1磷酸化其底物，包括丁胺卡那霉素和丁脲A的底物，传统上认为这是较差的底物酶。观察到磷酸转移酶活性的最佳pH为7.0-7.5。发现纯化的酶在不存在还原剂的情况下易于二聚。用胰蛋白酶处理酶，从N-末端切除了一个4kDa的片段，该片段含有氨基酸残基Cys-10。 27 kDa蛋白水解的APH（3'）-Ia不二聚，表明Cys-10通过二硫键参与二聚。分离出磷酸化的卡那霉素A，并确认磷酸化发生在3'-羟基处。此外，APH（3'）-Ia和APH（3'）-IIa均显示出使磷酸磷酸化的水（“ ATP水解酶”活性）的速率约为。比磷酸基转移至典型的氨基糖苷要慢10（4）-10（6）倍（对k（cat）/ K-m的影响）。产品抑制和替代底物诊断的结果表明APH（3'）-Ia氨基糖苷磷酸化的平衡-随机机制。

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