Glu-192----Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin.
In serine proteases, residue 192, three residues prior to the active site Ser-195, plays an important role in determining substrate specificity. In trypsin (EC 3.4.21.4) and most trypsin-like enzymes with relatively broad specificity, this position is occupied by Gln. In thrombin (EC 3.4.21.5), an enzyme with restricted specificity, position 192 is occupied by Glu. The potential importance of Glu-192 in restricting the specificity of thrombin was investigated by isosterically replacing Glu-192 with Gln. Unlike trypsin, thrombin cleavage of peptides with acidic residues in positions P3 and P'3 [where P3 and P'3 refer to three residues removed from the Arg (P1) cleavage site on the amino and carboxyl side, respectively] is inefficient. Protein C, an anticoagulant zymogen, has Asp residues in positions P3 and P'3. Thrombomodulin, an endothelial cell protein, complexes with thrombin to activate protein C rapidly thus altering the specificity of thrombin. Compared to thrombin, the Glu-192----Gln mutant thrombin activates protein C 22 times more rapidly and cleaves the P7-P'5 peptide from the protein C activation site 19 times faster. Enhanced protein C activation results primarily from an increase in the catalytic rate constant rather than an improved Michaelis constant, a property that is shared by the thrombin-thrombomodulin complex. The Glu-192----Gln mutation does not influence fibrinopeptide A release and only increases the rate of fibrinopeptide B release 2.7-fold. These results demonstrate that Glu-192 plays a critical role in restricting the specificity of thrombin and suggest that thrombomodulin may function in part by altering the enzyme-substrate interaction near residue 192 in thrombin.
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
B F
BF
Le Bonniec
C T
CT
Esmon
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D005971:Glutamates; D018698:Glutamic Acid; D005973:Glutamine; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011516:Prothrombin / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*
D005971:Glutamates; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D005973:Glutamine; D018698:Glutamic Acid; D011516:Prothrombin; D014357:Trypsin; D013917:Thrombin