McKay G A, Robinson R A, Lane W S, Wright G D
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Biochemistry. 1994 Nov 29;33(47):14115-20. doi: 10.1021/bi00251a021.
The aminoglycoside antibiotics are inactivated by modifying enzymes that are now widely distributed in many pathogenic bacteria. This situation threatens the continued use of these clinically important drugs. We have undertaken studies to understand the molecular mechanism of aminoglycoside resistance, and we report the affinity labeling of the enterococcal aminoglycoside 3'-phosphotransferase, APH(3')-IIIa, with an electrophilic ATP analogue, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). Incubation of purified APH(3')-IIIa with FSBA resulted in time-dependent irreversible inactivation of enzyme activity with a binding constant, Ki, of 0.406 mM and a rate of maximal inactivation, kmax, of 0.086 min-1. Addition of ATP completely protected the enzyme from inactivation, consistent with labeling of the ATP binding site. Reaction of APH(3')-IIIa with [14C]FSBA showed that inactivated APH(3')-IIIa incorporates 1 mol of FSBA/mol of enzyme. Peptide mapping of FSBA-inactivated APH(3')-IIIa resulted in the identification of two peptide peaks with highly increased absorbance at 260 nm, indicative of covalent labeling with FSBA. Analysis by electrospray ionization mass spectrometry and Edman degradation revealed two tryptic peptides, Val31-Lys44 and Leu34-Arg49, which incorporated the FSBA label at Lys33 and Lys44, respectively. This establishes the importance of the N-terminal region of APHs in ATP binding, a region of these enzymes which has heretofore not been considered for involvement in substrate binding.
氨基糖苷类抗生素会被修饰酶灭活，而这些修饰酶如今广泛分布于许多病原菌中。这种情况威胁到了这些临床重要药物的持续使用。我们开展了研究以了解氨基糖苷类耐药的分子机制，并报告了用亲电ATP类似物5'-[对-(氟磺酰基)苯甲酰基]腺苷(FSBA)对肠球菌氨基糖苷3'-磷酸转移酶APH(3')-IIIa进行亲和标记的情况。将纯化的APH(3')-IIIa与FSBA一起温育导致酶活性随时间不可逆失活，结合常数Ki为0.406 mM，最大失活速率kmax为0.086 min-1。添加ATP可完全保护该酶不被失活，这与ATP结合位点的标记情况一致。APH(3')-IIIa与[14C]FSBA的反应表明，失活的APH(3')-IIIa每摩尔酶结合1摩尔FSBA。对FSBA失活的APH(3')-IIIa进行肽图谱分析，结果鉴定出两个在260 nm处吸光度大幅增加的肽峰，表明与FSBA发生了共价标记。通过电喷雾电离质谱和埃德曼降解分析显示，有两个胰蛋白酶肽段，即Val31-Lys44和Leu34-Arg49，分别在Lys33和Lys44处结合了FSBA标记。这确立了APHs N端区域在ATP结合中的重要性，而此前一直未考虑该区域参与底物结合。
