Neutron and X-ray crystal structures of a perdeuterated enzyme inhibitor complex reveal the catalytic proton network of the Toho-1 ß-lactamase for the acylation reaction.
J Biol Chem
; 288(7): 4715-22, 2013 Feb 15.
Article
em En
| MEDLINE
| ID: mdl-23255594
ABSTRACT
The mechanism by which class A ß-lactamases hydrolyze ß-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 ß-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 ß-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 (11)B, as (10)B 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Beta-Lactamases
/
Acilação
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Proteínas de Escherichia coli
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Escherichia coli
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article