Linked Life Data

15075344

Source:http://linkedlifedata.com/resource/pubmed/id/15075344

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
27
pubmed:dateCreated
2004-6-28
pubmed:databankReference
pubmed:abstractText
Lipopolysaccharyl-alpha-1,4-galactosyltransferase C (LgtC), a glycosyltransferase family 8 alpha-1,4-galactosyltransferase from Neisseria meningitidis, catalyzes the transfer of galactose from UDP galactose to terminal lactose-containing acceptor sugars with net retention of anomeric configuration. To investigate the potential role of discrete nucleophilic catalysis suggested by the double displacement mechanism generally proposed for retaining glycosyltransferases, the side chain amide of Gln-189, which is suitably positioned to act as the catalytic nucleophile of LgtC, was substituted with the more nucleophilic carboxylate-containing side chain of glutamate in the hope of accumulating a glycosyl-enzyme intermediate. The resulting mutant was subjected to kinetic, mass spectrometric, and x-ray crystallographic analysis. Although the K(m) for UDP-galactose is not significantly altered, the k(cat) was reduced to 3% that of the wild type enzyme. Electrospray mass spectrometric analysis revealed that a steady state population of the Q189E variant contains a covalently bound galactosyl moiety. Liquid chromatographic/mass spectrometric analysis of fragmented proteolytic digests identified the site of labeling not as Glu-189 but, surprisingly, as the sequentially adjacent Asp-190. However, the side chain carboxylate of Asp-190 is located 8.9 A away from the donor substrate in the available crystal structure. Kinetic analysis of a D190N mutant at this position revealed a k(cat) value 3000-fold lower than that of the wild type enzyme. A 2.6-A crystal structure of the Q189E mutant with bound uridine 5'-diphospho-2-deoxy-2-fluoro-alpha-d-galactopyranose revealed no significant perturbation of the mode of donor sugar binding nor of active site configuration. This is the first trapping of an intermediate in the active site of a retaining glycosyltransferase and, although not conclusive, implicates Asp-190 as an alternative candidate catalytic nucleophile, thereby rekindling a longstanding mechanistic debate.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
2
pubmed:volume
279
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
28339-44
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:15075344-Aspartic Acid, pubmed-meshheading:15075344-Bacterial Proteins, pubmed-meshheading:15075344-Binding Sites, pubmed-meshheading:15075344-Catalysis, pubmed-meshheading:15075344-Chromatography, Liquid, pubmed-meshheading:15075344-Crystallography, X-Ray, pubmed-meshheading:15075344-Galactose, pubmed-meshheading:15075344-Glycosyltransferases, pubmed-meshheading:15075344-Kinetics, pubmed-meshheading:15075344-Lactose, pubmed-meshheading:15075344-Mass Spectrometry, pubmed-meshheading:15075344-Models, Chemical, pubmed-meshheading:15075344-Models, Molecular, pubmed-meshheading:15075344-Mutagenesis, Site-Directed, pubmed-meshheading:15075344-Mutation, pubmed-meshheading:15075344-Neisseria meningitidis, pubmed-meshheading:15075344-Peptides, pubmed-meshheading:15075344-Protein Structure, Tertiary, pubmed-meshheading:15075344-Spectrometry, Mass, Electrospray Ionization, pubmed-meshheading:15075344-Uridine Diphosphate Galactose
pubmed:year
2004
pubmed:articleTitle
Intermediate trapping on a mutant retaining alpha-galactosyltransferase identifies an unexpected aspartate residue.
pubmed:affiliation
Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't