McKay G A, Wright G D
Department of Biochemistry, McMaster University Hamilton, Ontario, Canada.
J Biol Chem. 1995 Oct 20;270(42):24686-92. doi: 10.1074/jbc.270.42.24686.
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 (Km(kanA)), whereas no inhibition is observed when the concentration of kanamycin A is held at 10Km(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 uncompetitive 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.
细菌对氨基糖苷 - 氨基环醇类抗生素的耐药性主要是由多种酶对药物进行共价修饰介导的。一种这样的修饰酶，即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，通过对抗生素上特定羟基残基进行ATP依赖性磷酸化作用，使多种氨基糖苷失活。通过使用终产物和产物抑制剂研究，我们首次对3'-氨基糖苷磷酸转移酶 - IIIa的动力学机制进行了详细研究。从稳态动力学推导的初始速度模式表明这是一种有序机制，首先是ATP有序结合，随后是氨基糖苷结合。AMP和腺苷酰亚胺二磷酸的终产物抑制对ATP而言是竞争性的，对卡那霉素A而言是非竞争性的。妥布霉素（一种缺乏3'-OH的卡那霉素类似物）的终产物抑制对卡那霉素A和ATP而言都是竞争性的，这表明底物是有序结合的，其中ATP必须在氨基糖苷添加之前添加。当卡那霉素A保持在亚饱和浓度（Km(kanA)）时，磷酸卡那霉素的产物抑制对ATP是非竞争性的，而当卡那霉素A的浓度保持在10Km(kanA)时则未观察到抑制作用。这与磷酸卡那霉素是首先释放的产物随后是ADP释放是一致的。观察到的抑制模式支持一种机制，即ATP结合先于氨基糖苷结合，随后是快速催化步骤。产物释放以有序方式进行，其中磷酸卡那霉素快速释放，随后是ADP的缓慢释放。氨基糖苷底物，如卡那霉素A，表现出对ATP而言是非竞争性的底物抑制。这表明在高药物浓度下氨基糖苷与缓慢解离的（E - ADP）复合物结合。这些实验与3'-氨基糖苷磷酸转移酶 - IIIa的Theorell - Chance动力学机制一致。
