Linked Life Data

8823158

Source:http://linkedlifedata.com/resource/pubmed/id/8823158

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
38
pubmed:dateCreated
1996-11-5
pubmed:abstractText
Two mutant beta-lactamases from Staphylococcus aureus PC1 which probe key catalytic residues have been produced by site-directed mutagenesis. In the S70A enzyme, the nucleophilic group that attacks the beta-lactam carbonyl carbon atom was eliminated. Consequently, the kcat values for hydrolysis of benzylpenicillin and nitrocefin have been reduced by 10(4)-10(5) compared with the wild-type enzyme. The crystal structure of S70A beta-lactamase has been determined at 2.1 A resolution. With the exception of the mutation site, the structure is identical to that of the native enzyme. The residual activity is attributed either to mistranslation that leads to production of wild-type enzyme and/or to remaining features of the active site that stabilize the tetrahedral transition state. Soaking of the crystals with ampicillin or clavulanate, followed by flash-freezing, has been carried out and the structures examined at 2.0 A resolution. For both experiments, the difference electron density maps revealed buildup of density in the active site that presumably corresponds to beta-lactam binding. However, neither electron density is sufficiently clear for defining the atomic details of the bound compounds. The K73H beta-lactamase has been prepared to test the possible role of Lys73 in proton transfer. It exhibits no detectable activity toward benzylpenicillin, and 10(5)-fold reduction of kcat for nitrocefin hydrolysis compared with the wild-type enzyme. No significant recovery of activity has been measured when the pH was varied between 5.0 and 8.0. The crystal structure of K73H beta-lactamase has been determined at 1.9 A resolution. While the overall structure is similar to that of the native enzyme, the electrostatic interactions between His73 and neighboring residues indicate that the imidazole ring is positively charged. In addition, the hydroxyl group of Ser70 adopts a position that is incompatible with nucleophilic attack on substrates. A crystal soaked with ampicillin was flash-frozen, and diffraction data were collected at 2.1 A resolution. The electron density map showed no indication of substrate binding.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
24
pubmed:volume
35
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
12251-8
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Structure and kinetics of the beta-lactamase mutants S70A and K73H from Staphylococcus aureus PC1.
pubmed:affiliation
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville 20850, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't