Linked Life Data

15134725

Source:http://linkedlifedata.com/resource/pubmed/id/15134725

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
6
pubmed:dateCreated
2004-5-11
pubmed:abstractText
The chromophoric spin-label substrate 6-N-[3-(2,2,5,5-tetramethyl-1-oxypyrrolin-3-yl)-propen-2-oyl]penicillanic acid (SLPPEN) was synthesized by acylation of 6-aminopenicillanic acid with the acid chloride of 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)-2-propenoic acid and characterized by physical methods. By application of angle-selected electron nuclear double resonance (ENDOR), we have determined the molecular structure of SLPPEN in solution. SLPPEN exhibited UV absorption properties that allowed accurate monitoring of the kinetics of its enzyme-catalyzed hydrolysis. The maximum value of the (substrate-product) difference extinction coefficient was 2824 M(-1) cm(-1) at 275 nm compared to 670 M(-1) cm(-1) at 232 nm for SLPEN [J. Am. Chem. Soc. 117 (1995) 6739]. For SLPPEN, the steady-state kinetic parameters kcat and kcat/KM, determined under initial velocity conditions, were 637 +/- 36 s(-1) and 13.8 +/- 1.4 x 10(6) M(-1) s(-1), respectively, for hydrolysis catalyzed by TEM-1 beta-lactamase of E. coli, and 0.5 +/- 0.04 s(-1) and 3.9 +/- 0.4 x 10(4) M(-1) s(-1) for hydrolysis catalyzed by the beta-lactamase of Enterobacter cloacae P99. We have also observed "burst kinetics" for the hydrolysis of SLPPEN with P99 beta-lactamase, indicative of formation of an acylenzyme reaction intermediate. In DMSO:H2O (30:70, v:v) cryosolvent mixtures buffered to pH* 7.0, the half-life of the acylenzyme intermediate formed with the P99 enzyme at -5 degrees C was > or = 3 min, suitable for optical characterization. The observation of burst kinetics in the hydrolysis of SLPPEN catalyzed by P99 beta-lactamase suggests that this chromophoric spin-labeled substrate is differentially sensitive to active site interactions underlying the cephalosporinase and penicillinase reactivity of this class C enzyme.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
1386-1425
pubmed:author
pubmed:issnType
Print
pubmed:volume
60
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1279-89
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:15134725-Acylation, pubmed-meshheading:15134725-Binding Sites, pubmed-meshheading:15134725-Buffers, pubmed-meshheading:15134725-Dimethyl Sulfoxide, pubmed-meshheading:15134725-Electron Spin Resonance Spectroscopy, pubmed-meshheading:15134725-Enterobacter cloacae, pubmed-meshheading:15134725-Escherichia coli, pubmed-meshheading:15134725-Freezing, pubmed-meshheading:15134725-Hydrogen-Ion Concentration, pubmed-meshheading:15134725-Hydrolysis, pubmed-meshheading:15134725-Kinetics, pubmed-meshheading:15134725-Models, Molecular, pubmed-meshheading:15134725-Molecular Structure, pubmed-meshheading:15134725-Penicillanic Acid, pubmed-meshheading:15134725-Protein Conformation, pubmed-meshheading:15134725-Sensitivity and Specificity, pubmed-meshheading:15134725-Solutions, pubmed-meshheading:15134725-Spin Labels, pubmed-meshheading:15134725-Structure-Activity Relationship, pubmed-meshheading:15134725-Substrate Specificity, pubmed-meshheading:15134725-Water, pubmed-meshheading:15134725-beta-Lactamases
pubmed:year
2004
pubmed:articleTitle
Chromophoric spin-labeled beta-lactam antibiotics for ENDOR structural characterization of reaction intermediates of class A and class C beta-lactamases.
pubmed:affiliation
Department of Biochemistry and Molecular Biology, Cummings Life Science Center, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. dmustafi@midway.uchicago.edu
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.