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Kinetic Evidence for a Second Ligand Binding Site on Streptococcus pneumoniae Penicillin-Binding Protein 2x
Biochemistry ( IF 2.9 ) Pub Date : 2018-02-27 00:00:00 , DOI: 10.1021/acs.biochem.7b01209 S. A. Adediran 1 , Kumar Subarno Sarkar 1 , R. F. Pratt 1
Biochemistry ( IF 2.9 ) Pub Date : 2018-02-27 00:00:00 , DOI: 10.1021/acs.biochem.7b01209 S. A. Adediran 1 , Kumar Subarno Sarkar 1 , R. F. Pratt 1
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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 PhCH2CONHCH2COSCH(D-Me)CO2– (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.
中文翻译：
肺炎链球菌青霉素结合蛋白2x上第二个配体结合位点的动力学证据
B类的高分子量青霉素结合蛋白（PBP，DD肽酶），例如肺炎链球菌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标。尽管它们在这方面很重要，但是由于缺少有用的底物，这些酶的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，而是在体内反应，但是某些酶（例如PBP2x）却催化某些硫酯的反应，例如PhCH 2 CONHCH 2 COSCH（D-Me）CO 2 –（2）。现在，我们已经制备了几种肽聚糖模拟的硫酯，我们希望它们与这些酶的天然底物更加相似。然而，令我们惊讶的是，尽管这些化合物确实是PBP2x的底物，但与2不同，它们似乎没有在水解过程中形成酰基酶中间体，并且它们的营业额受到某些肽和N-酰基氨基酸的抑制作用要弱得多。的2。这类抑制剂，N-苄氧羰基-d-谷氨酸，也淬灭了在6-肽半乳糖苷Openicillanic酸在DD-肽酶活性位点标记的PBP2x的荧光。这些结果是根据其中肽聚糖模拟的硫酯优先结合并在除经典DD-肽酶活性位点以外的位点处水解的模型来解释的。该第二位点可能代表体内结合肽聚糖底物或调节剂的延伸结合位点的一部分。这样的位点可能是未来抑制剂/抗生素设计的目标。
更新日期：2018-02-27
中文翻译：
肺炎链球菌青霉素结合蛋白2x上第二个配体结合位点的动力学证据
B类的高分子量青霉素结合蛋白（PBP，DD肽酶），例如肺炎链球菌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标。尽管它们在这方面很重要，但是由于缺少有用的底物，这些酶的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，而是在体内反应，但是某些酶（例如PBP2x）却催化某些硫酯的反应，例如PhCH 2 CONHCH 2 COSCH（D-Me）CO 2 –（2）。现在，我们已经制备了几种肽聚糖模拟的硫酯，我们希望它们与这些酶的天然底物更加相似。然而，令我们惊讶的是，尽管这些化合物确实是PBP2x的底物，但与2不同，它们似乎没有在水解过程中形成酰基酶中间体，并且它们的营业额受到某些肽和N-酰基氨基酸的抑制作用要弱得多。的2。这类抑制剂，N-苄氧羰基-d-谷氨酸，也淬灭了在6-肽半乳糖苷Openicillanic酸在DD-肽酶活性位点标记的PBP2x的荧光。这些结果是根据其中肽聚糖模拟的硫酯优先结合并在除经典DD-肽酶活性位点以外的位点处水解的模型来解释的。该第二位点可能代表体内结合肽聚糖底物或调节剂的延伸结合位点的一部分。这样的位点可能是未来抑制剂/抗生素设计的目标。
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