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Titolo:
CONVERTING BLOOD-COAGULATION FACTOR IXA INTO FACTOR XA - DRAMATIC INCREASE IN AMIDOLYTIC ACTIVITY IDENTIFIES IMPORTANT ACTIVE-SITE DETERMINANTS
Autore:
HOPFNER KP; BRANDSTETTER H; KARCHER A; KOPETZKI E; HUBER R; ENGH RA; BODE W;
Indirizzi:
MAX PLANCK INST BIOCHEM,ABT STRUKTURFORSCH D-82152 MARTINSRIED GERMANY BOEHRINGER MANNHEIM GMBH D-82372 PENZBERG GERMANY
Titolo Testata:
EMBO journal
fascicolo: 22, volume: 16, anno: 1997,
pagine: 6626 - 6635
SICI:
0261-4189(1997)16:22<6626:CBFIIF>2.0.ZU;2-A
Fonte:
ISI
Lingua:
ENG
Soggetto:
PLASMA THROMBOPLASTIN ANTECEDENT; BOVINE FACTOR-IX; CHRISTMAS-FACTOR; HUMAN-PROTHROMBIN; HEMOPHILIA-B; FACTOR-XIA; SUBSTRATE-SPECIFICITY; ESCHERICHIA-COLI; SERINE PROTEASES; RAY STRUCTURE;
Keywords:
BLOOD COAGULATION; FACTOR IX; IN VITRO FOLDING; SITE-DIRECTED MUTAGENESIS; SPECIFICITY;
Tipo documento:
Article
Natura:
Periodico
Citazioni:
47
Recensione:
Indirizzi per estratti:
Citazione:
K.P. Hopfner et al., "CONVERTING BLOOD-COAGULATION FACTOR IXA INTO FACTOR XA - DRAMATIC INCREASE IN AMIDOLYTIC ACTIVITY IDENTIFIES IMPORTANT ACTIVE-SITE DETERMINANTS", EMBO journal, 16(22), 1997, pp. 6626-6635

Abstract

The coagulation factors IXa (fIXa) and Xa (Ma) share extensive structural and functional homology; both cleave natural substrates effectively only with a cofactor at a phospholipid surface. However, the amidolytic activity of fIXa is 10(4)-fold lower than that of Ma. To identifydeterminants of this poor reactivity, we expressed variants of truncated fIXa (rf9a) and Ma (rf10a) in Escherichia coil. The crystal structures of fIXa and Ma revealed four characteristic active site components which were subsequently exchanged between rf9a and rf10a. ExchangingGlu219 by Gly or exchanging the 148 loop did not increase activity ofrf9a, whereas corresponding mutations abolished reactivity of rf10a. Exchanging Ile213 by Val only moderately increased reactivity of rf9a. Exchanging the 99 loop, however, dramatically increased reactivity. Furthermore, combining all four mutations essentially introduced Ma properties into rf9a: the amidolytic activity was increased 130-fold withMa substrate selectivity. The results suggest a 2-fold origin of fIXa's poor reactivity. A narrowed S3/S4 subsite disfavours interaction with substrate P3/P4 residues, while a distorted S1 subsite disfavours effective cleavage of the scissile bond. Both defects could be repairedby introducing Ma residues. Such engineered coagulation enzymes will be useful in diagnostics and in the development of therapeutics.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 18/09/20 alle ore 16:57:09