Linked Life Data

10441376

Source:http://linkedlifedata.com/resource/pubmed/id/10441376

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1999-9-9
pubmed:abstractText
A naturally occurring mutant of human thymidylate synthase (hTS) that contains a Tyr to His mutation at residue 33 was found to confer 4-fold resistance to 5-fluoro-2'-deoxyuridine (FdUrd), a prodrug of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The crystal structure of hTS implicated this Tyr residue in a drug resistance mechanistic role that may include both substrate binding and catalysis (Schiffer et al., Biochemistry, 34, 16279-16287, 1995). Because of the existence of a defined kinetic scheme and the development of a bacterial expression vector for the overproduction of Escherichia coli TS (ecTS), we chose to initially study the corresponding residue in the bacterial enzyme, Tyr 4 of ecTS. Nine mutant ecTS enzymes that differed in sequence at position 4 were generated. Mutants with a charged or polar side chain (Ser, Cys, Asp, and Arg) and Gly precipitated in the cell paste, resulting in no catalytic activity in cell-free extracts. Although most of the His 4 mutant precipitated, sufficient amounts remained in the cell-free extract to permit isolation to near homogeneity. Wild-type ecTS and mutants with a hydrophobic side chain (Phe, Ile, and Val) were expressed at nearly 30% of the total cellular protein. The k(cat) values for the isolatable mutants were 2- to 10-fold lower than that of the wild-type enzyme, while the K(m) values for 2'-deoxyuridylate (dUMP) and 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were similar for all the mutants. Dissociation constants for binary complex formation determined by stopped-flow spectroscopy were similar for the wild-type and mutant enzymes for both dUMP and 2'-deoxythymidylate, indicating that this mutation does not significantly alter the binding of the natural nucleotide ligands. However, each mutant enzyme had three- to 5-fold lower affinity for FdUMP in the binary complex compared with the wild-type enzyme, and only His 4 showed a lower affinity for FdUMP in the ternary complex. Analysis of k(burst) showed that the initial binding of CH(2)H(4)folate is weaker for each mutant compared to the wild-type enzyme and that lower k(cat) values were due to compromised rates that govern the chemical transformation of bound substrates to bound products.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0003-9861
pubmed:author
pubmed:copyrightInfo
Copyright 1999 Academic Press.
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
368
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
257-64
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Kinetic studies on drug-resistant variants of Escherichia coli thymidylate synthase: functional effects of amino acid substitutions at residue 4.
pubmed:affiliation
Department of Chemistry and Biochemistry, University of South Carolina, 730 South Main Street, Columbia, South Carolina, 29208, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't