期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 117, 期 11, 页码 5818-5825出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1922203117
关键词
serine beta-lactamase; avibactam; beta-lactamase inhibitor; acyl-enzyme; pKa perturbation
资金
- National Institutes of Health [AI103158, AI147654, GM129519, GM115854]
- NSF [CHE-1464946]
- NSF Graduate Research Fellowship Program [3900101301]
- US Department of Energy, Office of Biological and Environmental Research [DE-AC02-06CH11357]
- National Cancer Institute [ACB-12002]
- National Institute of General Medical Sciences [AGM-12006]
Gram-negative bacteria expressing class A beta-lactamases pose a serious health threat due to their ability to inactivate all beta-lactam antibiotics. The acyl-enzyme intermediate is a central milestone in the hydrolysis reaction catalyzed by these enzymes. However, the protonation states of the catalytic residues in this complex have never been fully analyzed experimentally due to inherent difficulties. To help unravel the ambiguity surrounding class A beta-lactamase catalysis, we have used ultrahigh-resolution X-ray crystallography and the recently approved beta-lactamase inhibitor avibactam to trap the acyl-enzyme complex of class A beta-lactamase CTX-M-14 at varying pHs. A 0.83-angstrom-resolution CTX-M-14 complex structure at pH 7.9 revealed a neutral state for both Lys73 and Glu166. Furthermore, the avibactam hydroxylamine-O-sulfonate group conformation varied according to pH, and this conformational switch appeared to correspond to a change in the Lys73 protonation state at low pH. In conjunction with computational analyses, our structures suggest that Lys73 has a perturbed acid dissociation constant (pK(a)) compared with acyl-enzyme complexes with beta-lactams, hindering its function to deprotonate Glu166 and the initiation of the deacylation reaction. Further NMR analysis demonstrated Lys73 pK(a) to be similar to 5.2 to 5.6. Together with previous ultrahigh-resolution crystal structures, these findings enable us to follow the proton transfer process of the entire acylation reaction and reveal the critical role of Lys73. They also shed light on the stability and reversibility of the avibactam carbamoyl acyl-enzyme complex, highlighting the effect of substrate functional groups in influencing the protonation states of catalytic residues and subsequently the progression of the reaction.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据