Linked Life Data

7028756

Source:http://linkedlifedata.com/resource/pubmed/id/7028756

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
23
pubmed:dateCreated
1982-1-28
pubmed:abstractText
Thymidylate synthetase of Saccharomyces cerevisiae was purified over 20,000-fold to apparent homogeneity by a procedure involving two new affinity methods and several precautions for avoiding proteolysis. Molecular weight of the native enzyme was about 65,000, as determined by gel filtration and velocity sedimentation. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band of molecular weight 30,000, suggesting that thymidylate synthetase is a dimer of very similar or identical subunits. The purified enzyme exhibited normal Michaelis-Menten kinetics toward both substrates, with apparent Km values for dUMP and for (--)-5,10-methylene-tetrahydropteroylglutamate of 5 microM and 70 microM, respectively. When the pentaglutamyl form of the cofactor was used, its apparent Km was lower (7 microM), but Vmax was unaltered. Reaction kinetics and product inhibition studies were most consistent with an ordered mechanism, wherein dUMP is the first substrate to bind and 7,8-dihydropteroylglutamate is the first product released. Halogenated analogs of the nucleotide substrate were competitive inhibitors of the yeast enzyme, with apparent Ki values for 5-fluoro-dUMP of 5 nM and for 5-Br-dUMP of 10 microM. Analogs of the cofactor were also competitive inhibitors, with apparent Ki values for both methotrexate and aminopterin of about 20 microM. Cibacron blue, a dye used as the ligand in an affinity adsorbent for one of the purification steps, was a potent competitive inhibitor with respect to either substrate, yielding apparent Ki values of 4 nM for the nucleotide binding site and 40 nM for the cofactor binding site.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
10
pubmed:volume
256
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
12456-62
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1981
pubmed:articleTitle
Thymidylate synthetase from Saccharomyces cerevisiae. Purification and enzymic properties.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.