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
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.