|
rdf:type |
|
|
lifeskim:mentions |
|
|
pubmed:issue |
1
|
|
pubmed:dateCreated |
2008-12-29
|
|
pubmed:abstractText |
Clavulanic acid is a widely used beta-lactamase inhibitor whose key beta-lactam core is formed by beta-lactam synthetase. beta-Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by beta-lactam formation. 32PPi-ATP exchange assays showed the first irreversible step of catalysis is acyl-adenylation. From a small, normal solvent isotope effect (1.38 +/- 0.04), it was concluded that beta-lactam synthesis contributes at least partially to kcat. Site-specific mutation of Lys-443 identified this residue as the ionizable group at pKa approximately 8.1 apparent in the pH-kcat profile that stabilizes the beta-lactam-forming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on kcat attenuating the observed solvent isotope effect on beta-lactam formation. Adherence to Kramers' theory gave a slope of 1.66 +/- 0.08 from a plot of log(o kcat/kcat) versus log(eta/eta(o)) consistent with opening of a structured loop visible in x-ray data preceding product release. Internal "friction" within the enzyme contributes to a slope of > 1 in this analysis. Correspondingly, earlier in the catalytic cycle ordering of a mobile active site loop upon substrate binding was manifested by an inverse solvent isotope effect (0.67 +/- 0.15) on kcat/Km. The increased second-order rate constant in heavy water was expected from ordering of this loop over the active site imposing torsional strain. Finally, an Eyring plot displayed a large enthalpic change accompanying loop movement (DeltaH approximately 20 kcal/mol) comparable to the chemical barrier of beta-lactam formation.
|
|
pubmed:grant |
|
|
pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-10422585,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-10506998,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-10869089,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-10969022,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-10985764,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-11302708,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-11601963,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-11844680,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-12409610,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-12413541,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-12458195,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-12679848,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-12820893,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-12890666,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-12962477,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-14700277,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-1547333,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-15700950,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-15835911,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-1615323,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-16530442,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-16716070,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-17287353,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-17355126,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-17658887,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-18062234,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-1883831,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-2012834,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-2018546,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-21524,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-2744487,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-3365378,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-3427057,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-3954986,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-4027237,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-427112,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-5339507,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-5911617,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-6294457,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-6324682,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-7074086,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-7176923,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-7176924,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-8563639,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-8663072,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-8679630,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-8939426,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-9100023,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-9230061,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-9281835,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-9336980,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-9485312,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-9689037,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-9748211,
http://linkedlifedata.com/resource/pubmed/commentcorrection/18955494-9748330
|
|
pubmed:language |
eng
|
|
pubmed:journal |
|
|
pubmed:citationSubset |
IM
|
|
pubmed:chemical |
|
|
pubmed:status |
MEDLINE
|
|
pubmed:month |
Jan
|
|
pubmed:issn |
0021-9258
|
|
pubmed:author |
|
|
pubmed:issnType |
Print
|
|
pubmed:day |
2
|
|
pubmed:volume |
284
|
|
pubmed:owner |
NLM
|
|
pubmed:authorsComplete |
Y
|
|
pubmed:pagination |
207-17
|
|
pubmed:dateRevised |
2010-9-21
|
|
pubmed:meshHeading |
|
|
pubmed:year |
2009
|
|
pubmed:articleTitle |
Dissection of the stepwise mechanism to beta-lactam formation and elucidation of a rate-determining conformational change in beta-lactam synthetase.
|
|
pubmed:affiliation |
Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
|
|
pubmed:publicationType |
Journal Article,
Research Support, N.I.H., Extramural
|
