Adediran S A, Sarkar Kumar Subarno, Pratt R F
Department of Chemistry , Wesleyan University , Lawn Avenue , Middletown , Connecticut 06459 , United States.
Biochemistry. 2018 Mar 20;57(11):1758-1766. doi: 10.1021/acs.biochem.7b01209. Epub 2018 Feb 27.
High molecular mass penicillin-binding proteins (PBPs, DD-peptidases) of class B, such as Streptococcus pneumoniae PBP2x, catalyze the cross-linking of peptidoglycan in bacterial cell wall biosynthesis and are thus important antibiotic targets. Despite their importance in this regard, structure-function studies of ligands of these enzymes have been impeded by the absence of useful substrates. In vitro, these enzymes do not catalyze peptide hydrolysis or aminolysis, their in vivo reaction, but some, such as PBP2x, do catalyze these reactions of certain thioesters such as PhCHCONHCHCOSCH(D-Me)CO (2). We have now prepared several peptidoglycan-mimetic thioesters that we expected to more closely resemble the natural substrates of these enzymes. To our surprise, however, these compounds, although indeed substrates of PBP2x, did not, unlike 2, appear to form an acyl-enzyme intermediate during hydrolysis, and their turnover was inhibited by certain peptides and N-acylamino acids much more weakly than that of 2. An inhibitor of this type, N-benzyloxycarbonyl-d-glutamic acid, also quenched the fluorescence of PBP2x that had been labeled at the DD-peptidase active site by 6-dansylamidopenicillanic acid. These results were interpreted in terms of a model where the peptidoglycan-mimetic thioesters preferentially bound to and hydrolyzed at a site other than the classical DD-peptidase active site. This second site is likely to represent part of an extended binding site that accommodates a peptidoglycan substrate or regulator in vivo. Such a site may be a target for future inhibitor/antibiotic design.
B类高分子量青霉素结合蛋白（PBPs，DD - 肽酶），如肺炎链球菌PBP2x，在细菌细胞壁生物合成中催化肽聚糖的交联，因此是重要的抗生素靶点。尽管它们在这方面很重要，但由于缺乏有用的底物，对这些酶配体的结构 - 功能研究受到了阻碍。在体外，这些酶不催化肽水解或氨解反应（它们在体内的反应），但一些酶，如PBP2x，确实能催化某些硫酯（如PhCHCONHCHCOSCH(D - Me)CO (2)）的这些反应。我们现已制备了几种肽聚糖模拟硫酯，预期它们能更接近这些酶的天然底物。然而，令我们惊讶的是，这些化合物虽然确实是PBP2x的底物，但与2不同，在水解过程中似乎没有形成酰基酶中间体，并且它们的周转受到某些肽和N - 酰基氨基酸的抑制程度比2弱得多。这种类型的抑制剂N - 苄氧羰基 - d - 谷氨酸，也能淬灭由6 - 丹磺酰胺青霉烷酸标记在DD - 肽酶活性位点的PBP2x的荧光。这些结果根据一个模型来解释，即肽聚糖模拟硫酯优先结合并在经典DD - 肽酶活性位点以外的位点水解。这个第二个位点可能代表一个扩展结合位点的一部分，该位点在体内容纳肽聚糖底物或调节剂。这样的位点可能是未来抑制剂/抗生素设计的靶点。
