Linked Life Data

9166793

Source:http://linkedlifedata.com/resource/pubmed/id/9166793

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
20
pubmed:dateCreated
1997-6-23
pubmed:databankReference
pubmed:abstractText
The possible role of the hydroxyl group of Tyr 108 in the catalytic mechanism of human glutathione transferase P1-1 has been investigated by means of site-directed mutagenesis, steady-state kinetic analysis, and crystallographic studies. Three representative cosubstrates have been used, i.e. ethacrynic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and 1-chloro-2,4-dinitrobenzene. In the presence of ethacrynic acid, the enzyme follows a rapid equilibrium random bi-bi mechanism with a rate-limiting step which occurs after the addition of the substrates and before the release of products. The replacement of Tyr 108 with Phe yields a 14-fold decrease of k(cat), while it does not change appreciably the affinity of the H site for the substrate. In this case, it would appear that the role of the hydroxyl function is to stabilize the transition state for the chemical step, i.e. the Michael addition of GSH to the electrophilic substrate. Crystallographic data are compatible with this conclusion showing the hydroxyl group of Y108 in hydrogen bonding distance of the ketone moiety of ethacrynic acid [Oakley, A. J., Rossjohn, J., Lo Bello, M., Caccuri, A. M., Federici, G., & Parker, M. W. (1997) Biochemistry 36, 576-585]. Moreover, no structural differences are observed between the Y108F mutant and the wild type, suggesting that the removal of the hydroxyl group is solely responsible for the loss of activity. A different involvement of Tyr 108 appears in the catalyzed conjugation of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole with GSH in which the rate-limiting step is of a physical nature, probably a structural transition of the ternary complex. The substitution of Tyr 108 yields an approximately 7-fold increase of k(cat) and a constant k(cat)/Km(NBD-Cl) value. Lack of a critical hydrogen bond between 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and Tyr 108 appears to be the basis of the increased k(cat). In the 1-chloro-2,4-dinitrobenzene/GSH system, no appreciable changes of kinetics parameters are found in the Y108F mutant. We conclude that Y108 has a multifunctional role in glutathione transferase P1-1 catalysis, depending on the nature of the electrophilic cosubstrate.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
20
pubmed:volume
36
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
6207-17
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:9166793-4-Chloro-7-nitrobenzofurazan, pubmed-meshheading:9166793-Crystallography, X-Ray, pubmed-meshheading:9166793-Dinitrochlorobenzene, pubmed-meshheading:9166793-Ethacrynic Acid, pubmed-meshheading:9166793-Glutathione S-Transferase pi, pubmed-meshheading:9166793-Glutathione Transferase, pubmed-meshheading:9166793-Humans, pubmed-meshheading:9166793-Isoenzymes, pubmed-meshheading:9166793-Kinetics, pubmed-meshheading:9166793-Metabolic Detoxication, Drug, pubmed-meshheading:9166793-Models, Molecular, pubmed-meshheading:9166793-Molecular Sequence Data, pubmed-meshheading:9166793-Mutagenesis, Site-Directed, pubmed-meshheading:9166793-Mutation, pubmed-meshheading:9166793-Recombinant Proteins, pubmed-meshheading:9166793-Substrate Specificity, pubmed-meshheading:9166793-Tyrosine, pubmed-meshheading:9166793-Viscosity
pubmed:year
1997
pubmed:articleTitle
Multifunctional role of Tyr 108 in the catalytic mechanism of human glutathione transferase P1-1. Crystallographic and kinetic studies on the Y108F mutant enzyme.
pubmed:affiliation
Department of Biology, University of Rome Tor Vergata, Italy.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't