Linked Life Data

12044179

Source:http://linkedlifedata.com/resource/pubmed/id/12044179

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
23
pubmed:dateCreated
2002-6-4
pubmed:abstractText
4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA to 4-hydroxybenzoyl-CoA by using an active site Asp145 carboxylate as the nucleophile. Formation of the corresponding Meisenheimer complex (EMc) is followed by chloride ion expulsion to form arylated enzyme (EAr). The EAr is then hydrolyzed to product. In this paper, we report the kinetics for dehalogenase-catalyzed 4-fluorobenzoyl-CoA (4-FBA-CoA) and 4-nitrobenzoyl-CoA (4-NBA-CoA) hydrolysis and provide Raman spectral evidence for the accumulation of EMc in these reactions. The 4-FBA-CoA and 4-NBA-CoA substrate analogues were selected for the poor leaving group ability of their C(4) substituents. Thus, the formation of the EAr from EMc should be hindered, giving rise to a quasi-steady-state equilibrium between EMc and the Michaelis complex. Detailed kinetic studies were carried out to quantitate the composition of the reaction mixtures. Quench experiments demonstrated that significant populations of EAr do not exist in reaction mixtures involving the 4-F- or 4-N-substrates. A kinetic model enabled us to estimate that approximately 10-20% of the enzyme-substrate complexes in the reaction mixtures are present as EMc. Raman difference spectra of 4-NBA-CoA and 4-FBA-CoA bound to WT and H90Q mutant dehalogenase have broad features near 1500 and 1220 cm(-1) that are absent in the free ligand. Crucially, these features are also absent in the Raman spectra of the complexes involving the D145A dehalogenase mutant that are unable to form an EMc. Quantum mechanical calculations, at the DFT level, provide strong support for assigning the novel 1500 and 1220 cm(-1) features to an EMc.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/4-chlorobenzoic acid, http://linkedlifedata.com/resource/pubmed/chemical/4-chlorobenzoyl coenzyme A, http://linkedlifedata.com/resource/pubmed/chemical/4-chlorobenzoyl coenzyme A..., http://linkedlifedata.com/resource/pubmed/chemical/4-fluorobenzoic acid, http://linkedlifedata.com/resource/pubmed/chemical/Acyl Coenzyme A, http://linkedlifedata.com/resource/pubmed/chemical/Alcohol Oxidoreductases, http://linkedlifedata.com/resource/pubmed/chemical/Bacterial Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Benzoic Acids, http://linkedlifedata.com/resource/pubmed/chemical/Chlorobenzoates, http://linkedlifedata.com/resource/pubmed/chemical/Hydrolases, http://linkedlifedata.com/resource/pubmed/chemical/NtnD protein, Pseudomonas sp. TW3
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
11
pubmed:volume
41
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7453-63
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Raman evidence for Meisenheimer complex formation in the hydrolysis reactions of 4-fluorobenzoyl- and 4-nitrobenzoyl-coenzyme A catalyzed by 4-chlorobenzoyl-coenzyme A dehalogenase.
pubmed:affiliation
Department of Chemistry, University of New Mexico, Albuquerque, NM 87131, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.