Linked Life Data

12823621

Source:http://linkedlifedata.com/resource/pubmed/id/12823621

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2003-6-25
pubmed:abstractText
A direct continuous UV-Vis spectrophotometric assay has been developed for VanX, a D-alanyl-D-alanine aminodipeptidase necessary for vancomycin resistance. This method is based on the hydrolysis of the alternative substrate D-alanyl-alpha-(R)-phenylthio-glycine D-Ala-D-Gly(S-Ph)-OH (H-DAla-DPsg-OH, 5a). Spontaneous decomposition of the released phenylthioglycine generates thiophenol, which is quantified using Ellman's reagent. The dipeptide behaved as an excellent substrate of VanX, exhibiting Michaelis-Menten kinetics with a kcat of 76 +/- 5/s and a KM of 0.83 +/- 0.08 mm (kcat = 46 +/- 3/s and KM = 0.11 +/- 0.01 mm for D-Ala-D-Ala). Determination of the kinetic parameters of the previously reported mechanism-based inhibitor D-Ala-D-Gly(SPhip-CHF2)-OH (H-D-Ala-DPfg-OH, 5c) [Araoz, R., Anhalt, E., René, L., Badet-Denisot, M.-A., Courvalin, P. & Badet, B. (2000) Biochemistry 39, 15971-15979] using the substrate reported in the present study yielded values of Kirr of 22 +/- 1 microM and kinact of 9.3 +/- 0.4/min in good agreement with values previously obtained in our laboratory (Kirr = 30 +/- 1 mm; kinact = 7.3 +/- 0.3/min). In addition, inhibition by the competing substrate D-Ala-D-Ala resulted in determination of a Ki = 70 +/- 6 microM close to the previously reported KM value. These results demonstrate that the present assay is a convenient, rapid and sensitive tool in the search for VanX inhibitors.
pubmed:commentsCorrections
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
1397-002X
pubmed:author
pubmed:issnType
Print
pubmed:volume
62
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
88-95
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
New chromogenic dipeptide substrate for continuous assay of the D-alanyl-D-alanine dipeptidase VanX required for high-level vancomycin resistance.
pubmed:affiliation
Institut de Chimie des Substances Naturelles, CNRS-UPR 2301, 91198 Gif-sur-Yvette, France.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't