Linked Life Data

16388585

Source:http://linkedlifedata.com/resource/pubmed/id/16388585

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2006-1-3
pubmed:abstractText
It is well established that electrostatic interactions play a vital role in enzyme catalysis. In this work, we report theory-guided mutation experiments that identified strong electrostatic contributions of a remote residue, namely, Glu232 located on the adjacent subunit, to 4-chlorobenzoyl-CoA dehalogenase catalysis. The Glu232Asp mutant was found to bind the substrate analogue 4-methylbenzoyl-CoA more tightly than does the wild-type dehalogenase. In contrast, the kcat for 4-chlorobenzoyl-CoA conversion to product was reduced 10000-fold in the mutant. UV difference spectra measured for the respective enzyme-ligand complexes revealed an approximately 3-fold shift in the equilibrium of the two active site conformers away from that inducing strong pi-electron polarization in the ligand benzoyl ring. Increased substrate binding, decreased ring polarization, and decreased catalytic efficiency indicated that the repositioning of the point charge in the Glu232Asp mutant might affect the orientation of the Asp145 carboxylate with respect to the substrate aromatic ring. The time course for formation and reaction of the arylated enzyme intermediate during a single turnover was measured for wild-type and Glu232Asp mutant dehalogenases. The accumulation of arylated enzyme in the wild-type dehalogenase was not observed in the mutant. This indicates that the reduced turnover rate in the mutant is the result of a slow arylation of Asp145, owing to decreased efficiency in substrate nucleophilic attack by Asp145. To rationalize the experimental observations, a theoretical model is proposed, which computes the potential of mean force for the nucleophilic aromatic substitution step using a hybrid quantum mechanical/molecular mechanical method. To this end, the removal or reorientation of the side chain charge of residue 232, modeled respectively by the Glu232Gln and Glu232Asp mutants, is shown to increase the rate-limiting energy barrier. The calculated 23.1 kcal/mol free energy barrier for formation of the Meisenheimer intermediate in the Glu232Asp mutant represents an increase of 6 kcal/mol relative to that of the wild-type enzyme, consistent with the 5.6 kcal/mol increase calculated from the difference in experimentally determined rate constants. On the basis of the combination of the experimental and theoretical evidence, we hypothesize that the Glu232(B) residue contributes to catalysis by providing an electrostatic force that acts on the Asp145 nucleophile.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
10
pubmed:volume
45
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
102-12
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed:year
2006
pubmed:articleTitle
Contributions of long-range electrostatic interactions to 4-chlorobenzoyl-CoA dehalogenase catalysis: a combined theoretical and experimental study.
pubmed:affiliation
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, Non-P.H.S., Research Support, N.I.H., Extramural