Identification of thrombin residues that modulate its interactions with antithrombin III and alpha 1-antitrypsin.
The role of thrombin's catalytic groove in the interaction with serpin has been investigated by comparing the association rate constant (kon) of several mutated thrombins with various serpins. The results indicated that Glu192, located three residues prior to the catalytic serine, and the major insertion in the sequence of thrombin compared with trypsin (residues Tyr60A-Trp60D) play an important role in modulating thrombin's interactions with serpins. Replacement of Glu192 by glutamine increased by 3 orders of magnitude the kon value with alpha 1-antitrypsin (which has a P1 methionine) but did not markedly alter the kon value with serpins containing a P1 arginine. The des-PPW thrombin mutant (lacking residues Pro60B, Pro60C, and Trp60D) exhibited a similar kon value as thrombin with protease nexin-1 but a kon 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 kon values for antithrombin III and protease nexin-1 with all thrombin variants tested, but a more dramatic effect was observed with a thrombin mutant (des-ETW) lacking residues Glu146, Thr147, and Trp148 (on the opposite side of the catalytic site relative to the 60-loop insertion). At the optimum concentration, heparin increased the kon value of the des-ETW--antithrombin III interaction by nearly 5 orders of magnitude, considerably 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.
Le Bonniec
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BF
Department of Haematology, University of Cambridge, England.
Guinto
E R
ER
Stone
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SR
D000595:Amino Acid Sequence; D000990:Antithrombin III / Q000378:metabolism*; D001665:Binding Sites; D004795:Enzyme Stability; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D016415:Sequence Alignment; D015842:Serine Proteinase Inhibitors / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism*; D017186:Titrimetry; D000515:alpha 1-Antitrypsin / Q000378:metabolism*
D015842:Serine Proteinase Inhibitors; D000515:alpha 1-Antitrypsin; D000990:Antithrombin III; D013917:Thrombin