Source:http://linkedlifedata.com/resource/pubmed/id/19736945
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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
41
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pubmed:dateCreated |
2009-10-13
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pubmed:abstractText |
Extended-spectrum beta-lactamases (ESBLs) are derivatives of enzymes such as SHV-1 and TEM-1 that have undergone site-specific mutations that enable them to hydrolyze, and thus inactivate, oxyimino-cephalosporins, such as cefotaxime and ceftazidime. X-ray crystallographic data provide an explanation for this in that the mutations bring about an expansion of the binding pocket by moving a beta-strand that forms part of the active site wall. Another characteristic of ESBLs that has remained enigmatic is the fact that they are "hypersusceptible" to inhibition by the mechanism-based inactivators tazobactam, sulbactam, and clavulanic acid. Here, we provide a rationale for this "hypersusceptibility" based on a comparative analysis of the intermediates formed by these compounds with wild-type (WT) SHV-1 beta-lactamase and its ESBL variants SHV-2 and SHV-5, which carry the G238S and G238S/E240K substitutions, respectively. A Raman spectroscopic analysis of the reactions in single crystals shows that, compared to WT, the SHV-2 and SHV-5 variants have relatively higher populations of the stable trans-enamine intermediate over the less stable and more easily hydrolyzable cis-enamine and imine co-intermediates. In solution, SHV-2 and SHV-5 also form larger populations of an enamine species compared to SHV-1 as detected by stopped-flow kinetic experiments under single-turnover conditions. Moreover, a simple Raman band shape analysis predicts that the trans-enamine intermediates themselves in SHV-2 and SHV-5 are held in more stable, rigid conformations compared to their trans-enamine analogues in WT SHV-1. As a result of this stabilization, more of the trans-enamine intermediate is formed, which subsequently lowers the K(I) values of the mechanism-based inhibitors up to 50-fold in SHV-2 and SHV-5.
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pubmed:grant | |
pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Cephalosporins,
http://linkedlifedata.com/resource/pubmed/chemical/Serine,
http://linkedlifedata.com/resource/pubmed/chemical/Sulbactam,
http://linkedlifedata.com/resource/pubmed/chemical/beta-Lactamases,
http://linkedlifedata.com/resource/pubmed/chemical/beta-lactamase SHV-2,
http://linkedlifedata.com/resource/pubmed/chemical/beta-lactamase SHV-5
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pubmed:status |
MEDLINE
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pubmed:month |
Oct
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pubmed:issn |
1520-4995
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pubmed:author | |
pubmed:issnType |
Electronic
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pubmed:day |
20
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pubmed:volume |
48
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
9912-20
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pubmed:dateRevised |
2011-9-26
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pubmed:meshHeading |
pubmed-meshheading:19736945-Catalytic Domain,
pubmed-meshheading:19736945-Cephalosporins,
pubmed-meshheading:19736945-Crystallography, X-Ray,
pubmed-meshheading:19736945-Kinetics,
pubmed-meshheading:19736945-Microbial Sensitivity Tests,
pubmed-meshheading:19736945-Mutagenesis, Site-Directed,
pubmed-meshheading:19736945-Plasmids,
pubmed-meshheading:19736945-Serine,
pubmed-meshheading:19736945-Spectrum Analysis, Raman,
pubmed-meshheading:19736945-Sulbactam,
pubmed-meshheading:19736945-beta-Lactamases
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pubmed:year |
2009
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pubmed:articleTitle |
Why the extended-spectrum beta-lactamases SHV-2 and SHV-5 are "hypersusceptible" to mechanism-based inhibitors.
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pubmed:affiliation |
Department of Biochemistry, Molecular Biology and Microbiology, and Medicine,Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, Non-P.H.S.,
Research Support, Non-U.S. Gov't,
Research Support, N.I.H., Extramural
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