期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 288, 期 7, 页码 4715-4722出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M112.436238
关键词
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资金
- Laboratory Directed Research and Development Program of Oak Ridge National Laboratory (ORNL)
- United States Department of Energy [DE-AC05-00OR22725]
- Scientific User Facilities Division, Office of Basic Energy Sciences, United States Department of Energy
- United States Department of Energy, Office of Biological and Environmental Research [DE-AC02-06CH11357]
The mechanism by which class A beta-lactamases hydrolyze beta-lactam antibiotics has been the subject of intensive investigation using many different experimental techniques. Here, we report on the novel use of both neutron and high resolution x-ray diffraction to help elucidate the identity of the catalytic base in the acylation part of the catalytic cycle, wherein the beta-lactam ring is opened and an acyl-enzyme intermediate forms. To generate protein crystals optimized for neutron diffraction, we produced a perdeuterated form of the Toho-1 beta-lactamase R274N/R276N mutant. Protein perdeuteration, which involves replacing all of the hydrogen atoms in a protein with deuterium, gives a much stronger signal in neutron diffraction and enables the positions of individual deuterium atoms to be located. We also synthesized a perdeuterated acylation transition state analog, benzothiophene-2-boronic acid, which was also isotopically enriched with B-11, as B-10 is a known neutron absorber. Using the neutron diffraction data from the perdeuterated enzyme-inhibitor complex, we were able to determine the positions of deuterium atoms in the active site directly rather than by inference. The neutron diffraction results, along with supporting bond-length analysis from high resolution x-ray diffraction, strongly suggest that Glu-166 acts as the general base during the acylation reaction.
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