Role of the Ω-loop in the activity, substrate specificity, and structure of class A β-lactamase
Role of the Ω-loop in the activity, substrate specificity, and structure of class A β-lactamase
复制标题
DOI：
10.1021/bi972127f
复制
发表时间：
1998-03-10
期刊：
影响因子：
2.9
通讯作者：
Herzberg, O
中科院分区：
文献类型：
--
作者：
Banerjee, S;Pieper, U;Herzberg, O
A类β-内酰胺酶的结构包含与活性位点相关的Ω环，其携带关键催化残基Glu 166。产生来自金黄色葡萄球菌PCl的β-内酰胺酶的16个残基Ω环缺失突变体，包括残基163-178，以检查环的功能和结构作用。晶体结构在2.3埃下测定和精制，并且用多种β-内酰胺抗生素表征突变酶的动力学。通常，野生型β-内酰胺酶水解青霉素化合物的效果优于头孢菌素。相比之下，Omega环的缺失导致仅作用于头孢菌素类（包括第三代化合物）的变体酶。动力学测量和电喷雾质谱分析表明，第一代和第三代头孢菌素形成稳定的酰基-酶复合物，除了显色头孢菌素，头孢硝肟，其酰化后的酶进行水解以1000倍慢于野生型β-内酰胺酶的速率。酰基-酶加合物的水解被阻止，因为Omega环的缺失消除了包含Glu 166及其相关亲核水位点的脱酰化装置。晶体结构显示，虽然突变酶的整体折叠与天然β-内酰胺酶相似，但在缺失环附近发生了局部调整。β-内酰胺特异性的改变归因于这些结构变化。在天然结构中，Ω环限制活性位点凹陷边缘处的β链构象。环的去除为β链提供了新程度的构象柔性，使得其向内朝向活性位点空间移位。模拟的米氏复合物与苄青霉素和头孢噻啶表明，β-链的扰动构象与青霉素结合不一致，因为β-内酰胺侧链取代基和β-链之间的空间冲突。相反，头孢菌素结合时不发生冲突。第三代头孢菌素的识别是可能的，因为这些化合物典型的β-内酰胺环的大体积侧链取代基可以容纳在通过缺失Ω环而释放的空间中。
The structure of class A beta-lactamases contains an Omega-loop associated with the active site, which carries a key catalytic residue, Glu166. A 16-residue Omega-loop deletion mutant of beta-lactamase from Staphylococcus aureus PCl, encompassing residues 163-178, was produced in order to examine the functional and structural role of the loop. The crystal structure was determined and refined at 2.3 Angstrom, and the kinetics of the mutant enzyme was characterized with a variety of beta-lactam antibiotics. In general, the wild-type beta-lactamase hydrolyzes penicillin compounds better than cephalosporins. In contrast, the deletion of the Omega-loop led to a variant enzyme that acts only on cephalosporins, including third generation compounds. Kinetic measurements and electrospray mass spectrometry revealed that the first and third generation cephalosporins form stable acyl-enzyme complexes, except for the chromogenic cephalosporin, nitrocefin, which after acylating the enzyme undergoes hydrolysis at a 1000-fold slower rate than that with wild-type beta-lactamase. Hydrolysis of the acyl-enzyme adducts is prevented because the deletion of the Omega-loop eliminates the deacylation apparatus comprising Glu166 and its associated nucleophilic water site. The crystal structure reveals that while the overall fold of the mutant enzyme is similar to that of the native beta-lactamase, local adjustments in the vicinity of the missing loop occurred. The altered beta-lactam specificity is attributed to these structural changes. In the native structure, the Omega-loop restricts the conformation of a beta-strand at the edge of the active site depression. Removal of the loop provides the beta-strand with a new degree of conformational flexibility, such that it is displaced inward toward the active site space. Modeled Michaelis complexes with benzylpenicillin and cephaloridine show that the perturbed conformation of the beta-strand is inconsistent with penicillin binding because of steric clashes between the beta-lactam side chain substituent and the beta-strand. In contrast, no clashes occur upon cephalosporin binding. Recognition of third generation cephalosporins is possible because the bulky side chain substituents of the beta-lactam ring typical of these compounds can be accommodated in the space freed by the deletion of the Omega-loop.