Linked Life Data

9585520

Source:http://linkedlifedata.com/resource/pubmed/id/9585520

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
20
pubmed:dateCreated
1998-6-22
pubmed:abstractText
Structural analyses of bacterial thymidylate synthases (TSs) implicate a serine residue corresponding to Ser216 in human TS in hydrogen bond networks that are involved in binding of the nucleotide substrate, 2'-deoxyuridylate (dUMP), and that stabilize a beta-bulge in the protein. Utilizing site-directed mutagenesis, 12 mutant proteins were created with substitutions at residue 216. DNA complementation studies utilizing a TS-negative bacterial strain revealed that only one mutant, Thr216 TS, supports the growth of the bacteria in the absence of thymidine. Kinetic characterization of the mutant proteins revealed that all TSs except Thr216 TS exhibited kcat/Kms for dUMP that are 10(3)-10(4) times lower, relative to that of wild-type TS. In addition, Thr216 TS was the only mutant to bind the mechanism-based inhibitor, 5-fluoro-2'-deoxyuridylate (FdUMP), into a ternary complex. Ligand binding studies revealed that Kds for dUMP binding to two defective mutants, Ala216 and Leu216 TSs, are 12-16-fold higher than that of wild-type TS. The data are consistent with the hypothesis that serine at this relative position is involved in dUMP binding; however, the data indicate that Ser216 has effects on catalysis, in addition to effects on dUMP binding. Catalysis is initiated by nucleophilic attack of the active site cysteine of TS on dUMP. The reaction rates of cysteine residues with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) were slower for Ala216 TS than for wild-type TS.
pubmed:grant
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
19
pubmed:volume
37
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7096-102
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1998
pubmed:articleTitle
A hydroxyl group at residue 216 is essential for catalysis by human thymidylate synthase.
pubmed:affiliation
Department of Chemistry, University of South Carolina, Columbia 29208, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.