Kinetic mechanism of metallo-β-lactamase L1 from Stenotrophomonas maltophilia
Kinetic mechanism of metallo-β-lactamase L1 from Stenotrophomonas maltophilia
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DOI：
10.1021/bi9826512
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发表时间：
1999-02-02
期刊：
影响因子：
2.9
通讯作者：
Crowder, MW
中科院分区：
文献类型：
--
作者：
McManus-Munoz, S;Crowder, MW
采用快速扫描和停流紫外-可见（UV-vis）研究了头孢硝肟与嗜麦芽窄食单胞菌金属β-内酰胺酶L1的反应，以探讨L1水解青霉素和头孢菌素的动力学机制。通过L1对头孢硝肟水解的快速扫描和停流紫外-可见光谱研究，鉴定了三种物质：（1）基材头孢硝肟在390 nm处有吸收峰（λ = 11 500 M-1 cm（-1）），在反应过程中以170 +/- 30 s（-1）的速率常数降低（2）产物（水解的硝头孢菌素）在485 nm处显示吸收峰（λ = 17 420 M-1 cm（-1）），在反应过程中增加，速率常数为40 +/- 1 s（-1）;以及（3）中间体在665 nm处显示吸收峰（λ = 32 000 M-1 cm（-1）），最初以190 +/- 3 s（-1）的速率常数增加，然后以38 +/- 2 s（-1）的速率常数减少.单周转实验表明，有没有前稳态的爆发与头孢硝肟的反应，此外，进步曲线可以拟合的动力学机制，其中包括通过使用KINSIM和上面给出的速率常数的瞬态中间体的形成。在不同的反应条件下或与不同的基板进行的实验的进展曲线也可以适合所提出的动力学机制。中间体的存在沿着动力学模拟的证据支持L1的水解机制，其涉及中间体的分解是速率决定的。
The reaction of nitrocefin with metallo-beta-lactamase L1 from Stenotrophomonas maltophilia was studied using rapid-scan and stopped-flow ultraviolet-visible (UV-vis) studies in an effort to discern the kinetic mechanism used by L1 to hydrolyze penicillins and cephalosporins. Rapid-scan and stopped-flow UV-vis studies of nitrocefin hydrolysis by L1 identified three species: (1) the substrate (nitrocefin) displayed an absorbance peak at 390 nm (epsilon = 11 500 M-1 cm(-1)) that decreased during the reaction with a rate constant of 170 +/- 30 s(-1) (2) the product (hydrolyzed nitrocefin) displayed an absorbance peak at 485 nm (epsilon = 17 420 M-1 cm(-1)) that increased during the reaction with rate constant of 40 +/- 1 s(-1); and (3) an intermediate displayed an absorbance peak at 665 nm (epsilon = 32 000 M-1 cm(-1)) that increased initially with a rate constant of 190 +/- 3 s(-1) and then decreased with a rate constant of 38 +/- 2 s(-1). Single-turnover experiments demonstrated that there were no pre-steady-state bursts in the reaction of L1 with nitrocefin; moreover, the progress curves could be fit to a kinetic mechanism that includes the formation of a transient intermediate by using KINSIM and the rate constants given above. Progress curves from experiments conducted at different reaction conditions or with a different substrate could also be fit to the proposed kinetic mechanism. The evidence for the presence of an intermediate along with kinetic simulations supports a hydrolytic mechanism for L1 that involves an intermediate whose breakdown is rate-determining.