Linked Life Data

15494418

Source:http://linkedlifedata.com/resource/pubmed/id/15494418

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
52
pubmed:dateCreated
2004-12-21
pubmed:abstractText
Thrombin formation results from cleavage of prothrombin following Arg(271) and Arg(320). Both bonds are accessible for cleavage, yet the sequential action of prothrombinase on Arg(320) followed by Arg(271) is implied by the intermediate observed during prothrombin activation. We have studied the individual cleavage reactions catalyzed by prothrombinase by using a series of recombinant derivatives: wild type prothrombin (II(WT)) contained both cleavage sites; II(Q271) contained a single cleavable site at Arg(320); II(Q320) and II(A320) contained a single cleavable site at Arg(271); and II(QQ) was resistant to cleavage. Cleavage at Arg(320) in II(Q271) could account for the initial cleavage reaction leading to the consumption of either plasma prothrombin or II(WT), whereas cleavage at Arg(271) in either II(Q320) or II(A320) was found to be approximately 30-fold slower. Equivalent kinetic constants were obtained for three of the four possible half-reactions. Slow cleavage at Arg(271) in intact prothrombin resulted from an approximately 30-fold reduction in V(max). Thus, the observed pathway of bond cleavage by prothrombinase can be explained by the kinetic constants for the four possible individual cleavage reactions. II(Q320) was a competitive inhibitor of II(Q271) cleavage, and II(QQ) was a competitive inhibitor for each reaction with K(i) approximately K(m). The data are inconsistent with previous proposals and suggest a model in which substrates for each of the four possible half-reactions bind in a mutually exclusive manner and with equal affinity to prothrombinase in a cleavage site-independent way. Despite equivalent exosite binding interactions between all four possible substrates and the enzyme, we propose that ordered bond cleavage results from the constraints associated with the binding of substrates in one of two conformations to a single form of prothrombinase.
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
24
pubmed:volume
279
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
54927-36
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:15494418-Amino Acid Sequence, pubmed-meshheading:15494418-Arginine, pubmed-meshheading:15494418-Binding Sites, pubmed-meshheading:15494418-Cell Line, pubmed-meshheading:15494418-Cloning, Molecular, pubmed-meshheading:15494418-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:15494418-Humans, pubmed-meshheading:15494418-Kinetics, pubmed-meshheading:15494418-Mutagenesis, Insertional, pubmed-meshheading:15494418-Peptide Fragments, pubmed-meshheading:15494418-Protein Binding, pubmed-meshheading:15494418-Protein Conformation, pubmed-meshheading:15494418-Prothrombin, pubmed-meshheading:15494418-Recombinant Proteins, pubmed-meshheading:15494418-Structure-Activity Relationship, pubmed-meshheading:15494418-Substrate Specificity, pubmed-meshheading:15494418-Thromboplastin, pubmed-meshheading:15494418-Transfection
pubmed:year
2004
pubmed:articleTitle
Binding of substrate in two conformations to human prothrombinase drives consecutive cleavage at two sites in prothrombin.
pubmed:affiliation
Joseph Stokes Research Institute, Children's Hospital of Philadelphia, University of Pennsylvania, PA 19104, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.