IDENTIFICATION OF THROMBIN RESIDUES THAT MODULATE ITS INTERACTIONS WITH ANTITHROMBIN-III AND ALPHA-1-ANTITRYPSIN

Citation
Bf. Lebonniec et al., IDENTIFICATION OF THROMBIN RESIDUES THAT MODULATE ITS INTERACTIONS WITH ANTITHROMBIN-III AND ALPHA-1-ANTITRYPSIN, Biochemistry, 34(38), 1995, pp. 12241-12248
Citations number
68
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
34
Issue
38
Year of publication
1995
Pages
12241 - 12248
Database
ISI
SICI code
0006-2960(1995)34:38<12241:IOTRTM>2.0.ZU;2-1
Abstract
The role of thrombin's catalytic groove in the interaction with serpin has been investigated by comparing the association rate constant (k(o n)) of several mutated thrombins with various serpins. The results ind icated that Glu(192), located three residues prior to the catalytic se rine, and the major insertion in the sequence of thrombin compared wit h trypsin (residues Tyr(60A)-Trp(60D)) play an important role in modul ating thrombin's interactions with serpins. Replacement of Glu(192) by glutamine increased by 3 orders of magnitude the k(on) value with alp ha 1-antitrypsin (which has a P-1 methionine) but did not markedly alt er the k(on) value with serpins containing a P-1 arginine. The des-PPW thrombin mutant (lacking residues pro(60B), Pro(60C), and Trp(60D)) e xhibited a similar k(on) value as thrombin with protease nexin-1 but a k(on) value 2 orders of magnitude lower with antithrombin III. Thus, the 60-loop insertion of thrombin appears critical for its interaction with antithrombin III but dispensable for the formation of a complex with protease nexin-1. Heparin increased markedly the k(on) values for antithrombin III and protease nexin-1 with all thrombin variants test ed, but a more dramatic effect was observed with a thrombin mutant (de s-ETW) lacking residues Glu(146), Thr(147), and Trp(148) (on the oppos ite side of the catalytic site relative to the 60-loop insertion). At the optimum concentration, heparin increased the k(on) value of the de s-ETW-antithrombin III interaction by nearly 5 orders of magnitude, co nsiderably more than for thrombin, suggesting that heparin is able to compensate in part for the adverse effects of the des-ETW mutation on the structure of thrombin.