KINETIC MECHANISM OF AMINOGLYCOSIDE PHOSPHOTRANSFERASE TYPE IIIA - EVIDENCE FOR A THEORELL-CHANCE MECHANISM
KINETIC MECHANISM OF AMINOGLYCOSIDE PHOSPHOTRANSFERASE TYPE IIIA - EVIDENCE FOR A THEORELL-CHANCE MECHANISM
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DOI：
10.1074/jbc.270.42.24686
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发表时间：
1995-10-20
影响因子：
4.8
通讯作者：
WRIGHT, GD
中科院分区：
文献类型：
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作者：
MCKAY, GA;WRIGHT, GD
细菌对氨基糖苷-氨基环醇类抗生素的耐药性主要是通过多种酶对药物进行共价修饰介导的。其中一种修饰酶，3'-氨基糖苷磷酸转移酶，由革兰氏阳性球菌（如肠球菌和链球菌）产生，通过atp依赖性磷酸化抗生素上的特定羟基残基，使多种氨基糖苷失活。通过使用终端和产物抑制剂的研究，我们首次详细研究了3'-氨基糖苷磷酸转移酶iiia的动力学机制，从稳态动力学推断的初始速度模式表明了一个顺序的机制，首先是ATP的有序结合，然后是氨基糖苷的有序结合。AMP和腺苷酰-咪多二磷酸的终端抑制作用与ATP竞争，与卡那霉素a竞争，与卡那霉素a无竞争，与卡那霉素a无竞争，与卡那霉素a无竞争，缺乏3'-羟基卡那霉素类似物的托布霉素的终端抑制作用与卡那霉素a竞争，与ATP无竞争，表明有序底物结合，ATP必须先加入氨基糖苷，当卡那霉素a保持在亚饱和浓度（K-m(kanA)）时，卡那霉素磷酸的产物抑制作用与ATP无竞争。而当卡那霉素A的浓度保持在10 K-m（kanA）时，没有观察到抑制作用。这与磷酸卡那霉素是第一个释放的产物，其次是ADP释放一致。观察到的抑制模式支持ATP结合先于氨基糖苷结合的机制，然后是快速的催化步骤，产物释放以有序的方式进行，其中磷酸卡那霉素快速释放，随后缓慢释放ADP。氨基糖苷底物，如卡那霉素A，对底物的抑制作用与ATP不具有竞争性。这表明氨基糖苷与缓慢解离的(E。ADP)复合物在高药物浓度下。这些实验符合3′-氨基糖苷磷酸转移酶iiia的Theorell-Chance动力学机制。
Bacterial resistance to aminoglycoside-aminocyclitol antibiotics is mediated primarily by covalent modification of the drugs by a variety of enzymes. One such modifying enzyme, the 3'-aminoglycoside phosphotransferase, which is produced by Gram-positive cocci such as Enterococcus and Streptococcus inactivates a broad range of aminoglycosides by ATP-dependent phosphorylation of specific hydroxyl residues on the antibiotics. Through the use of dead-end and product inhibitor studies, we present the first detailed examination of the kinetic mechanism for the 3'-aminoglycoside phosphotransferase-IIIa, Initial velocity patterns deduced from steady-state kinetics indicate a sequential mechanism with ordered binding of ATP first followed by aminoglycoside. Dead-end inhibition by AMP and adenylyl-imidodiphosphate is competitive versus ATP and noncompetitive versus kanamycin A. Dead-end inhibition by tobramycin, a kanamycin analogue lacking a 3'-OH, is competitive versus both kanamycin A and uncompetitive versus ATP, indicative of ordered substrate binding where ATP must add prior to aminoglycoside addition, Product inhibition by kanamycin phosphate is noncompetitive versus ATP when kanamycin A is held at subsaturating concentrations (K-m(kanA)), whereas no inhibition is observed when the concentration of kanamycin A is held at 10 K-m(kanA). This is consistent with kanamycin phosphate being the first product released followed by ADP release. The patterns of inhibition observed support a mechanism where ATP binding precedes aminoglycoside binding followed by a rapid catalytic step, Product release proceeds in an ordered fashion where kanamycin phosphate is released quickly followed by a slow release of ADP. Aminoglycoside substrates, such as kanamycin A, show substrate inhibition that is uncom petitive versus ATP. This indicates binding of the aminoglycosides to the slowly dissociating (E . ADP) complex at high drug concentrations. These experiments are consistent with a Theorell-Chance kinetic mechanism for 3'-aminoglycoside phosphotransferase-IIIa.