Relations between beta-lactamases and penicillin-binding proteins: beta-lactamase activity of penicillin-binding protein 5 from Escherichia coli.
Relations between beta-lactamases and penicillin-binding proteins: beta-lactamase activity of penicillin-binding protein 5 from Escherichia coli.
复制标题
β-内酰胺酶和青霉素结合蛋白之间的关系：大肠杆菌青霉素结合蛋白 5 的 β-内酰胺酶活性。
DOI：
10.1093/clinids/10.4.733
复制
发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Strominger,JL
中科院分区：
文献类型：
--
作者：
Nicholas,RA;Strominger,JL
研究了青霉素结合蛋白(PBP)5水解青霉素的分子机理。半胱氨酸-115以前被认为与PBP5中的酰基酶中间体的水解有关。在寡核苷酸定向突变的研究中，通过将半胱氨酸-115残基突变为丝氨酸或丙氨酸残基来研究半胱氨酸-115在PBP5机制中的作用。研究的蛋白质在其羧基末端的基因截断导致这些蛋白质的一种可溶形式(SPBPs)的产生。对纯化后的PBPs进行了β-内酰胺酶和丙氨酸羧肽酶活性测定。这些检测结果表明，半胱氨酸残基并不直接参与酶的作用机制。在进一步研究β-内酰胺水解酶的催化机理时，从聚乙二醇水溶液中大量纯化和结晶了一种可溶的、自然产生的突变体。PBP5的这个突变体(sPBP5‘)对[14C]青霉素具有接近正常的酰化速率，但显示出酰基-酶复合体的半衰期急剧增加。SPbP5‘晶体的衍射率为>；3？，适用于详细的结晶学分析。
The molecular mechanism of the hydrolysis of penicillin by penicillin-binding protein (PBP) 5 was investigated. Cysteine-115 had been previously implicated in the hydrolysis of the acyl-enzymeintermediate in PBP5. In studies with oligonucleotide-directed mutagenesis, the role of cysteine-115 in the mechanism of PBP5 was investigated by the mutation of this residue to either a serine or an alanine residue. Genetic truncation of the studied proteins at their carboxyl termini resulted in the production of a soluble form of these proteins (sPBPs). The purified sPBPs were assayed for both their β-lactamase and theird-alanine carboxypeptidase activities. The results of these assays indicate that the cysteine residue does not directly participate in the enzymatic mechanism. In a further investigation of the catalytic mechanism of β-lactam hydrolysis, a soluble, naturally occurring mutant was purified in large amounts and crystallized from concentrated polyethylene glycol solutions. This mutant of PBP5 (sPBP5') has a near-normal acylation rate with respect to [14C]penicillin, but shows a drastic increase in the half-life of the acyl-enzyme complex. The crystals of sPBP5' diffract to >3 Å resolution and are suitable for detailed crystallographic analysis.