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Titolo:
HEPARIN ENHANCES THE CATALYTIC ACTIVITY OF DES-ETW-THROMBIN
Autore:
GOODWIN CA; DEADMAN JJ; LEBONNIEC BF; ELGENDY S; KAKKAR VV; SCULLY MF;
Indirizzi:
THROMBOSIS RES INST,EMMANUEL KAYE BLDG,MANRESA RD LONDON SW3 6LR ENGLAND THROMBOSIS RES INST LONDON SW3 6LR ENGLAND UNIV CAMBRIDGE,CTR MRC,DEPT HAEMATOL CAMBRIDGE CB2 2QH ENGLAND
Titolo Testata:
Biochemical journal
, volume: 315, anno: 1996,
parte:, 1
pagine: 77 - 83
SICI:
0264-6021(1996)315:<77:HETCAO>2.0.ZU;2-#
Fonte:
ISI
Lingua:
ENG
Soggetto:
HUMAN ALPHA-THROMBIN; ANTITHROMBIN-III; PENTOSAN POLYSULFATE; BLOOD-COAGULATION; COFACTOR-II; BINDING; HIRUDIN; INHIBITORS; PEPTIDE; SITE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
55
Recensione:
Indirizzi per estratti:
Citazione:
C.A. Goodwin et al., "HEPARIN ENHANCES THE CATALYTIC ACTIVITY OF DES-ETW-THROMBIN", Biochemical journal, 315, 1996, pp. 77-83

Abstract

The thrombin mutant, des-ETW-thrombin, lacking Glu(146) Thr(147) and Trp(148) within a unique insertion loop located at the extreme end of the primary specificity pocket, has been shown previously to exhibit reduced catalytic activity with respect to macromolecular and syntheticthrombin substrates and reduced or enhanced susceptibility to inhibition. Investigation of the hydrolysis of peptidyl p-nitroanilide substrates by des-ETW-thrombin showed increased activity in the presence of heparin and other sulphated glycosaminoglycans. No effect was observedupon the activity of wild-type thrombin. Heparin was found to decrease the K-m for cleavage of four thrombin-specific substrates by des-ETW-thrombin, by 3-4-fold. Similarly, pentosan polysulphate (PPS) decreased the K-m with these substrates by 8-10-fold. Heparin also increased the rate of inhibition of des-ETW-thrombin by antithrombin III and D-phenylalanyl-prolylarginylchloromethane (PPACK). The inhibition of des-ETW-thrombin by a number of thrombin-specific peptide boronic acids also showed significant reduction in the final K-i in the presence of heparin, due to reduction in the off-rate. A peptide analogue of a sequence of hirudin which binds thrombin tightly to exosite 1 (fibrinogen recognition site) potentiated the activity of des-ETW-thrombin against peptide p-nitroanilide substrates in a manner similar to heparin. The K-i for the inhibition of des-ETW-thrombin by p-aminobenzamidine was decreased by these ligands from 9.7 mM to 7.5 mM, 5.1 mM and 2.5 mM in the presence of heparin, hirudin peptide and PPS respectively, suggesting the increased catalytic activity is due to enhanced access to the primary specificity pocket. The positive influence of these ligands ondes-ETW-thrombin was reversed in the presence of ATP or ADP; the latter has previously been shown to inhibit thrombin activity by blocking initial interaction with fibrinogen at exosite 1. Because the effect of heparin and PPS is similar to that of hirudin peptide, it is proposed that the most likely mechanism is that binding at the heparin-binding site (thrombin exosite 2) facilitates interaction at exosite I causing a conformational change which partially corrects the defective ground-state binding of the mutant thrombin. Although no effect was observed upon the activity of wild-type thrombin, our findings do provide further evidence of an allosteric property of thrombin which may controlthe geometry of, and access to, the primary specificity pocket.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 21/09/20 alle ore 00:56:45