当前位置： SCI文献检索 > BIOCHEMISTRY期刊下所有文献 > Elucidation of the structural basis for the slow reactivity of thrombin with plasminogen activator inhibitor-1.

Elucidation of the structural basis for the slow reactivity of thrombin with plasminogen activator inhibitor-1.

Abstract：

:Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor of the serpin superfamily which rapidly inactivates tissue plasminogen activator (tPA), but reacts with thrombin at a much slower rate. Based on the previous mutagenesis studies and the X-ray crystal structure of the thrombin E192Q-bovine pancreatic trypsin inhibitor (BPTI) complex, the structural basis for the slow reactivity of thrombin with PAI-1 is investigated in this study. In the crystal structure of the thrombin E192Q-BPTI complex, the reactive site loop of BPTI is stabilized in a canonical conformation by several productive interactions (e.g., Glu39 of thrombin is ion-paired to the P5' Arg, and Gln192 is hydrogen-bonded to the P2 and P4 backbone carbonyls of BPTI). PAI-1 contains Glu residues at both the P4' and P5' positions, and previous mutagenesis studies suggest that these residues make productive interactions with Arg39 of tPA as well as with two other positively charged residues present on the 39-loop of the protease (chymotrypsin numbering). Glu39 and Glu192 of thrombin would be unable to make such productive interactions with PAI-1. Instead, their repulsive interactions with the similarly charged residues and/or the backbone carbonyls of the PAI-1 reactive site loop could restrict the reaction. To test this, the rate constants (k2) for the PAI-1 inactivation of wild-type, E39K, E39Q, E192Q, E192M, and E39K/E192Q thrombins were determined. The inactivation rates of E39K [k2 = (4.3 +/- 0.2) x 10(4) M-1 s-1] and E39Q [k2 = (1.0 +/- 0.1) x 10(4) M-1 s-1] were 50- and 12-fold faster than the inactivation of wild-type thrombin [k2 = (8.6 +/- 0. 5) x 10(2) M-1 s-1], respectively. Relative to thrombin, the PAI-1 inactivation rates were improved 31-fold for E192Q [k2 = (2.7 +/- 0. 5) x 10(4) M-1 s-1] and 5-fold for E192M [k2 = (4.3 +/- 0.8) x 10(3) M-1 s-1] thrombins. With the double mutant E39K/E192Q, the inactivation rate [k2 = (5.4 +/- 0.4) x 10(5) M-1 s-1] was improved 628-fold over wild-type thrombin. These results suggest that repulsive interactions and/or lack of productive electrostatic interactions between PAI-1 and Glu39 and Glu192 of thrombin are responsible for the slow reaction of thrombin with this serpin.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Rezaie AR

doi

10.1021/bi9808518

subject

Has Abstract

pub_date

1998-09-22 00:00:00

pages

13138-42

issue

eissn

0006-2960

issn

1520-4995

pii

bi9808518

journal_volume

pub_type

杂志文章

在线工具

Elucidation of the structural basis for the slow reactivity of thrombin with plasminogen activator inhibitor-1.