Linked Life Data

8294436

Source:http://linkedlifedata.com/resource/pubmed/id/8294436

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
1994-2-25
pubmed:abstractText
Human thymidylate synthase is a polymeric protein composed of two subunits with identical primary structures. In this study we determined the binding affinities of 5,10-methylene tetrahydropteroyltetraglutamate (folate substrate) and a group of close structural folate analog inhibitors. Thymidylate synthase bound both mono and polyglutamylated folate substrates and analogs more tightly in the presence of deoxyuridylate. These results and product inhibition studies confirmed that the orders of substrate addition and product release from thymidylate synthase were similar for mono and polyglutamylated substrates. Equilibrium dialysis studies showed that the folate substrate in a ternary complex with deoxyuridylate bound to one of the subunits (site A) with a Kd of 720 nM. The binding of the substrate to the second subunit (site B) was much weaker, and the Kd could not be determined by this method. However, dissociation constants for each subunit could be measured for the folate analog inhibitors, and, depending on the inhibitor, the relative Kd value for each subunit varied substantially. For example, formyl-5,8-dideazafolate and tetraglutamylated 10-propargyl-5,8-dideazafolate bound to both sites with similar Kd values, whereas D1694Glu4 bound to subunit A with a higher affinity (Kd = 1.0 nM) than to subunit B (Kd = 30 nM). In contrast, 1843U89 (mono or diglutamylated form) had a much higher affinity for subunit B (Kd approximately 0.1 nM) compared with subunit A (Kd approximately 400 nM). Enzyme inhibition kinetic analyses showed that the Ki values of 1843U89 were quite low (0.1 nM) and that the inhibition was noncompetitive. In contrast, the other folate analogs inhibited the enzyme via mixed inhibition (i.e. both the Km for the folate substrate and the Vmax were altered). We conclude that the two subunits of thymidylate synthase bind folate substrates and analogs differently and that the asymmetric binding of the ligands is the major factor that determines the inhibition kinetics of each folate analog inhibitor.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/1843U89, http://linkedlifedata.com/resource/pubmed/chemical/2'-deoxyuridylic acid, http://linkedlifedata.com/resource/pubmed/chemical/CB 3717, http://linkedlifedata.com/resource/pubmed/chemical/Carbon Radioisotopes, http://linkedlifedata.com/resource/pubmed/chemical/Deoxyuracil Nucleotides, http://linkedlifedata.com/resource/pubmed/chemical/Folic Acid, http://linkedlifedata.com/resource/pubmed/chemical/Folic Acid Antagonists, http://linkedlifedata.com/resource/pubmed/chemical/Indoles, http://linkedlifedata.com/resource/pubmed/chemical/Isoindoles, http://linkedlifedata.com/resource/pubmed/chemical/Macromolecular Substances, http://linkedlifedata.com/resource/pubmed/chemical/Quinazolines, http://linkedlifedata.com/resource/pubmed/chemical/Thymidylate Synthase
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
21
pubmed:volume
269
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1873-82
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
1994
pubmed:articleTitle
Mode of binding of folate analogs to thymidylate synthase. Evidence for two asymmetric but interactive substrate binding sites.
pubmed:affiliation
Division of Molecular Genetics and Microbiology, Burroughs Wellcome Company, Research Triangle Park, North Carolina 27709.
pubmed:publicationType
Journal Article