Linked Life Data

11380262

Source:http://linkedlifedata.com/resource/pubmed/id/11380262

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
22
pubmed:dateCreated
2001-5-30
pubmed:abstractText
Heparin has been proposed to conformationally activate the serpin, antithrombin, by making the reactive center loop P1 arginine residue accessible to proteinases. To evaluate this proposal, we determined the effect of mutating the P1 arginine on antithrombin's specificity for target and nontarget proteinases in both native and heparin-activated states of the serpin. As expected, mutation of the P1 arginine to tryptophan, histidine, leucine, and methionine converted the specificity of antithrombin from a trypsin inhibitor (k(assoc) = 2 x 10(5) M(-1) s(-1)) to a chymotrypsin inhibitor (k(assoc) = 10(3)-10(5) M(-1) s(-1)). However, heparin pentasaccharide activation increased the reactivity of the P1 variants with chymotrypsin or of the wild-type inhibitor with trypsin only 2-6-fold, implying that the P1 residue had similar accessibilities to these proteinases in native and activated states. Mutation of the P1 arginine greatly reduced k(assoc) for antithrombin inhibition of thrombin and factor Xa from 40- to 5000-fold, but heparin normally accelerated the reactions of the variant antithrombins with these enzymes to make them reasonably efficient inhibitors (k(assoc) = 10(3)-10(4) M(-1) s(-1)). Fluorescence difference spectra of wild-type and P1 tryptophan variant antithrombins showed that the P1 tryptophan exhibited fluorescence properties characteristic of a solvent-exposed residue which were insignificantly affected by heparin activation. Moreover, all P1 variant antithrombins bound heparin with approximately 2-3-fold higher affinities than the wild type. These findings are consistent with the P1 mutations disrupting a P1 arginine-serpin body interaction which stabilizes the native low-heparin affinity conformation, but suggest that this interaction is of low energy and unlikely to limit the accessibility of the P1 residue. Together, these findings suggest that the P1 arginine residue is similarly accessible to proteinases in both native and heparin-activated states of the serpin and contributes similarly to the specificity of antithrombin for thrombin and factor Xa in the two serpin conformational states. Consequently, determinants other than the P1 residue are responsible for enhancing the specificity of antithrombin for the two proteinases when activated by heparin.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
5
pubmed:volume
40
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
6670-9
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:11380262-Animals, pubmed-meshheading:11380262-Antithrombins, pubmed-meshheading:11380262-Arginine, pubmed-meshheading:11380262-Binding Sites, pubmed-meshheading:11380262-Cell Line, pubmed-meshheading:11380262-Chymotrypsin, pubmed-meshheading:11380262-Cricetinae, pubmed-meshheading:11380262-Enzyme Activation, pubmed-meshheading:11380262-Factor Xa, pubmed-meshheading:11380262-Heparin, pubmed-meshheading:11380262-Humans, pubmed-meshheading:11380262-Mutagenesis, Site-Directed, pubmed-meshheading:11380262-Protein Conformation, pubmed-meshheading:11380262-Recombinant Proteins, pubmed-meshheading:11380262-Serine Proteinase Inhibitors, pubmed-meshheading:11380262-Serpins, pubmed-meshheading:11380262-Spectrometry, Fluorescence, pubmed-meshheading:11380262-Substrate Specificity, pubmed-meshheading:11380262-Thrombin
pubmed:year
2001
pubmed:articleTitle
The antithrombin P1 residue is important for target proteinase specificity but not for heparin activation of the serpin. Characterization of P1 antithrombin variants with altered proteinase specificity but normal heparin activation.
pubmed:affiliation
Center for Molecular Biology of Oral Diseases, University of Illinois at Chicago, 60612, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.