pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5
pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5
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DOI：
10.1016/s0167-4838(02)00311-4
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发表时间：
2002-06-03
期刊：
影响因子：
--
通讯作者：
Gutheil, WG
中科院分区：
文献类型：
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作者：
Stefanova, ME;Davies, C;Gutheil, WG
最近对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。研究了PBP 5的稳定性、线性反应动力学、酰基供体底物特异性、一些活性位点导向试剂的抑制作用和pH曲线。PBP 5表现出长达数小时的线性反应动力学。PBP 5的稀释通常导致活性的实质性损失，除非将BSA或BSA衍生物加入稀释缓冲液中。PBP 5没有表现出对一组简单的五种α-和ε-取代的L-Lys-D-Ala-D-Ala衍生物的显著偏好，表明PBP 5缺乏对细胞壁底物的交联状态的特异性。在许多活性位点导向试剂中，只有一些巯基导向试剂具有显著的抑制作用。值得注意的是，丝氨酸导向的试剂，有机磷酸盐，和简单的硼酸作为抑制剂是无效的。PBP 5在pH 4.6- 12.3范围内稳定，并且针对Ac-2-L-Lys-D-Ala-D-Ala的活性的k（cat）/K-m相对于pH的曲线为钟形，pK（a）s为8.2和11.1。这是第一个完整的pH值配置文件，包括酸性和碱性的肢体，为PBP催化的DD-羧肽酶（CPase）反应。根据其结构、与A类β-内酰胺酶的相似性以及诱变研究结果，将PBP 5 pH曲线的酸性和碱性分支分别归属于Lys-47和Lys-213。这一分配支持了赖氨酸-47作为酰化和去酰化反应的一般基础的作用。(C)2002 Elsevier Science B.V保留所有权利。
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 alpha- and epsilon-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 k(cat)/K-m vs. pH profile for activity against Ac-2-L-Lys-D-Ala-D-Ala was bell-shaped, with pK(a)s 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 beta-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. (C) 2002 Elsevier Science B.V All rights reserved.