Site-directed mutagenesis of glutamate 166 in two beta-lactamases - Kinetic and molecular modeling studies
Site-directed mutagenesis of glutamate 166 in two beta-lactamases - Kinetic and molecular modeling studies
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DOI：
10.1074/jbc.272.9.5438
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发表时间：
1997-02-28
影响因子：
4.8
通讯作者：
Frere, JM
中科院分区：
文献类型：
--
作者：
Guillaume, G;Vanhove, M;Frere, JM
A 类 β-内酰胺酶的催化途径涉及酰基酶中间体，其中底物与 Ser-70 残基进行酯连接，Glu-166 和 Lys-73 已被提议作为一般碱在丝氨酸 OH 基团激活中的候选者，TEM-1 中的天冬酰胺和白色链霉菌 G β-内酰胺酶中的组氨酸取代 Glu-166，产生形成的酶尽管苄青霉素对修饰蛋白的酰化作用仍然相对较快，但与用野生型酶观察到的相比，酰化作用明显受损。此外，E166N 取代导致底物谱发生了惊人的改变，远大于 Omega 环残基的其他突变所描述的改变。分子模型研究表明，催化水分子的位移可能与这一观察结果有关。这些结果证实了 Glu-166 和“催化”母体分子在酰化和脱酰化过程中的关键作用。
The catalytic pathway of class A beta-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue, Glu-166 and Lys-73 have been proposed as candidates for the role of general base in the activation of the serine OH group, The replacement of Glu-166 by an asparagine in the TEM-1 and by a histidine in the Streptomyces albus G beta-lactamases yielded enzymes forming stable acyl-enzymes with beta-lactam antibiotics, Although acylation of the modified proteins by benzylpenicillin remained relatively fast, it was significantly impaired when compared to that observed with the wild-type enzyme. Moreover, the E166N substitution resulted in a spectacular modification of the substrate profile much larger than that described for other mutations of Omega-loop residues. Molecular modeling studies indicate that the displacement of the catalytic water molecule can be related to this observation, These results confirm the crucial roles of Glu-166 and of the ''catalytic'' mater molecule in both the acylation and the deacylation processes.