Saves I, Burlet-Schiltz O, Maveyraud L, Samama J P, Promé J C, Masson J M
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, Centre National de la Recherche Scientifique, Toulouse, France.
Biochemistry. 1995 Sep 19;34(37):11660-7. doi: 10.1021/bi00037a003.
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 Km 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.
在TEM-1β-内酰胺酶的超广谱天然突变体中发现的G238S替换诱导了一种新的水解头孢噻肟的能力，并且对TEM-1的良好底物的活性大幅丧失。为了在分子水平上理解这一现象，通过将电喷雾质谱与紫外分光光度法相结合，开发了一种测定酰化和脱酰化基本速率常数的方法。TEM-1和G238S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行为非常不同。对于这两种酶，青霉素水解的限速步骤是脱酰化，而头孢噻肟水解的限速步骤是酰化。对G238S突变体的进一步分析表明，对青霉素活性的丧失是由于脱酰化速率大幅降低，而对头孢噻肟催化效率的提高是更好的米氏常数和酰化速率增加的结果。G238S突变体中基本速率常数和水解能力的这些改变可能与活性位点中ω-环构象的结构效应有关。
