pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5

Miglena E. Stefanova , Christopher Davies , Robert A. Nicholas , William G. Gutheil
{"title":"pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5","authors":"Miglena E. Stefanova ,&nbsp;Christopher Davies ,&nbsp;Robert A. Nicholas ,&nbsp;William G. Gutheil","doi":"10.1016/S0167-4838(02)00311-4","DOIUrl":null,"url":null,"abstract":"<div><p>The recent structural determination of <em>Escherichia coli</em> penicillin-binding protein 5 (PBP 5) provides the opportunity for detailed structure–function studies of this enzyme. PBP 5 was investigated in terms of its stability, linear reaction kinetics, acyl-donor substrate specificity, inhibition by a number of active site-directed reagents, and pH profile. PBP 5 demonstrated linear reaction kinetics for up to several hours. Dilution of PBP 5 generally resulted in substantial loss of activity, unless BSA or a BSA derivative was added to the diluting buffer. PBP 5 did not demonstrate a significant preference against a simple set of five α- and ε-substituted <span>l</span>-Lys-<span>d</span>-Ala-<span>d</span>-Ala derivatives, suggesting that PBP 5 lacks specificity for the cross-linked state of cell wall substrates. Among a number of active site-directed reagents, only some thiol-directed reagents gave substantial inhibition. Notably, serine-directed reagents, organic phosphates, and simple boronic acids were ineffective as inhibitors. PBP 5 was stable over the pH range 4.6–12.3, and the <em>k</em><sub>cat</sub>/<em>K</em><sub>m</sub> vs. pH profile for activity against Ac<sub>2</sub>-<span>l</span>-Lys-<span>d</span>-Ala-<span>d</span>-Ala was bell-shaped, with p<em>K</em><sub>a</sub>s at 8.2 and 11.1. This is the first complete pH profile, including both acidic and basic limbs, for a PBP-catalyzed <span>dd</span>-carboxypeptidase (CPase) reaction. Based on its structure, similarity to Class A β-lactamases, and results from mutagenesis studies, the acidic and basic limbs of the pH profile of PBP 5 are assigned to Lys-47 and Lys-213, respectively. This assignment supports a role for Lys-47 as the general base for acylation and deacylation reactions.</p></div>","PeriodicalId":100166,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2002-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0167-4838(02)00311-4","citationCount":"38","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0167483802003114","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 38

Abstract

The recent structural determination of Escherichia coli penicillin-binding protein 5 (PBP 5) provides the opportunity for detailed structure–function studies of this enzyme. PBP 5 was investigated in terms of its stability, linear reaction kinetics, acyl-donor substrate specificity, inhibition by a number of active site-directed reagents, and pH profile. PBP 5 demonstrated linear reaction kinetics for up to several hours. Dilution of PBP 5 generally resulted in substantial loss of activity, unless BSA or a BSA derivative was added to the diluting buffer. PBP 5 did not demonstrate a significant preference against a simple set of five α- and ε-substituted l-Lys-d-Ala-d-Ala derivatives, suggesting that PBP 5 lacks specificity for the cross-linked state of cell wall substrates. Among a number of active site-directed reagents, only some thiol-directed reagents gave substantial inhibition. Notably, serine-directed reagents, organic phosphates, and simple boronic acids were ineffective as inhibitors. PBP 5 was stable over the pH range 4.6–12.3, and the kcat/Km vs. pH profile for activity against Ac2-l-Lys-d-Ala-d-Ala was bell-shaped, with pKas at 8.2 and 11.1. This is the first complete pH profile, including both acidic and basic limbs, for a PBP-catalyzed dd-carboxypeptidase (CPase) reaction. Based on its structure, similarity to Class A β-lactamases, and results from mutagenesis studies, the acidic and basic limbs of the pH profile of PBP 5 are assigned to Lys-47 and Lys-213, respectively. This assignment supports a role for Lys-47 as the general base for acylation and deacylation reactions.

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
大肠杆菌青霉素结合蛋白5的pH值、抑制剂和底物特异性研究
最近大肠杆菌青霉素结合蛋白5 (PBP 5)的结构测定为该酶的详细结构功能研究提供了机会。对PBP 5的稳定性、线性反应动力学、酰基供体底物特异性、多种活性位点导向试剂的抑制作用和pH谱进行了研究。PBP 5表现出长达数小时的线性反应动力学。除非在稀释缓冲液中加入牛血清白蛋白或牛血清白蛋白衍生物,否则PBP - 5的稀释通常会导致活性的大量丧失。PBP 5对一组简单的α-和ε-取代的l-Lys-d-Ala-d-Ala衍生物没有表现出明显的偏好,这表明PBP 5对细胞壁底物的交联状态缺乏特异性。在许多活性位点导向试剂中,只有一些巯基导向试剂具有明显的抑制作用。值得注意的是,丝氨酸导向试剂、有机磷酸盐和简单硼酸作为抑制剂是无效的。PBP 5在4.6-12.3的pH范围内保持稳定,对Ac2-l-Lys-d-Ala-d-Ala活性的kcat/Km与pH曲线呈钟形,pKas分别为8.2和11.1。这是pbp催化的dd-羧基肽酶(CPase)反应的第一个完整的pH谱,包括酸性和碱性分支。基于其结构、与A类β-内酰胺酶的相似性以及诱变研究结果,PBP 5 pH谱的酸性和碱性分支分别归属于Lys-47和Lys-213。这种分配支持Lys-47作为酰化和去酰化反应的一般碱的作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
自引率
0.00%
发文量
0
期刊最新文献
High-molecular-weight protein hydrodynamics studied with a long-lifetime metal-ligand complex Structural basis for development of cathepsin B-specific noncovalent-type inhibitor: crystal structure of cathepsin B–E64c complex The role of β-strand 5A of plasminogen activator inhibitor-1 in regulation of its latency transition and inhibitory activity by vitronectin Yeast cytochrome c peroxidase: mechanistic studies via protein engineering Butyrylcholinesterase-catalyzed hydrolysis of N-methylindoxyl acetate: analysis of volume changes upon reaction and hysteretic behavior
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1