Molecular basis of residue 192 participation in determination of coagulation protease specificity

Residue 192 (chymotrypsin numbering system) in thrombin, activated protein C, and factor Xa contributes to the specificity of these enzymes toward their substrates and inhibitors. A Glu192-->Gln mutation in both thrombin and activated protein C yielded enzymes that reacted better with some, but not all, of their natural substrates and inhibitors. To determine whether the specificity change is due to productive interactions of Gln192 with substrates and inhibitors or elimination of repulsive electrostatic interactions, we prepared forms of thrombin, des-(1-45)-factor Xa and activated des-(1-45)-protein C with Glu, Gln, or Met at position 192 and compared their activities toward inhibitors and substrates. All mutants had nearly normal amidolytic activity. The Glu192-->Gln and Glu 192-->Met mutations of thrombin and activated des-(1-45)-protein C increased the second-order rate constant (k2) of inhibition by alpha 1-antitrypsin about 700-fold and 170-fold for thrombin, and 185-fold and 150-fold for activated des-(1-45)-protein C, respectively. [E192]faxtor Xa, but not [M192]factor Xa, was resistant to inhibition by alpha 1-antitrypsin. Glu-->Gln or Glu-->Met mutants of both thrombin and activated des-(1-45)-protein C were effectively inhibited by tissue factor pathway inhibitor (K1 < 200 nM) and, except for [M192]thrombin, by bovine pancreatic trypsin inhibitor (K1 < 60 nM). With respect to substrate cleavage, Glu192-->Gln and Glu192-->Met mutations of activated des-(1-45)-protein C both inactivated factor Va 2-3-fold faster than activated des-(1-45)-protein C. Thrombin and [M192]thrombin activated protein C at similar slow rates compared to rapid activation by [Q192]thrombin. The Gln192-->Met mutants of des-(1-45)-factor Xa activated prethrombin 1.8-11-fold slower than wild-type enzyme. With thrombomodulin or factor Va present, these differences in protein C and prethrombin 1 activation rates were decreased to about 2-fold. We conclude that residue 192 contribution to enzyme specificity is achieved by both productive and repulsive interactions and that the magnitude and nature of the participation varies among enzymes, substrates and inhibitors.