MASS-SPECTRAL KINETIC-STUDY OF ACYLATION AND DEACYLATION DURING THE HYDROLYSIS OF PENICILLINS AND CEFOTAXIME BY BETA-LACTAMASE TEM-1 AND THE G238S MUTANT
MASS-SPECTRAL KINETIC-STUDY OF ACYLATION AND DEACYLATION DURING THE HYDROLYSIS OF PENICILLINS AND CEFOTAXIME BY BETA-LACTAMASE TEM-1 AND THE G238S MUTANT
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DOI：
10.1021/bi00037a003
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发表时间：
1995-09-19
期刊：
影响因子：
2.9
通讯作者：
MASSON, JM
中科院分区：
文献类型：
--
作者：
SAVES, I;BURLETSCHILTZ, O;MASSON, JM
在TEM-1 β-内酰胺酶的超广谱天然突变体中发现的G238 S取代诱导了新的水解头孢噻肟的能力和对TEM-1良好底物的活性的大量损失。为了在分子水平上理解这一现象，已经开发了一种方法来确定酰化和脱酰化的基本速率常数，通过使用电喷雾质谱结合紫外分光光度法。TEM-1和G238 S突变体对青霉素和头孢噻肟的水解表明，青霉素和头孢噻肟的水解行为是非常不同的。对于两种酶，青霉素水解的限制性步骤是脱酰，而头孢噻肟水解的限制性步骤是酰化。对G238 S突变体的进一步分析表明，对青霉素的活性损失是由于脱酰速率的大幅降低，而对头孢噻肟的催化效率的增加是更好的K-m和增加的酰化速率的结果。这些修改的基本速率常数和水解能力的G238 S突变体可以链接到结构上的Ω环构象的活性位点的影响。
The G238S substitution found in extended-spectrum natural mutants of TEM-1 beta-lactamase induces a new capacity to hydrolyze cefotaxime and a large loss of activity against the good substrates of TEM-1. To understand this phenomenon at the molecular level, a method to determine the acylation and deacylation elementary rate constants has been developed by using electrospray mass spectrometry combined with UV spectrophotometry. The hydrolysis of penicillins and cefotaxime by TEM-1 and the G238S mutant shows that the behavior of penicillins and cefotaxime is very different. With both enzymes, the limiting step is deacylation for penicillin hydrolysis, but acylation for cefotaxime hydrolysis. Further analyses of the G238S mutant show that the loss of activity against penicillins is due to a large decrease in the deacylation rate and that the increase in catalytic efficiency against cefotaxime is the result of a better K-m and an increased acylation rate. These modifications of the elementary rate constants and the hydrolytic capacity in the G238S mutant could be linked to structural effects on the Omega-loop conformation in the active site.