INHIBITORY MECHANISM OF SERPINS - INTERACTION OF THROMBIN WITH ANTITHROMBIN AND PROTEASE NEXIN-1

The mechanism for the inhibition of thrombin by the serpins antithrombin and protease nexin 1 has been investigated using several kinetic techniques at pH 7.9 and 37 degrees C with an ionic strength of 0.3 M. Rapid kinetic studies demonstrated that a two-step mechanism for the formation of the stable thrombin-serpin complex applied to both serpins. The inhibition constant for the initial thrombin-antithrombin complex was 265 mu M, and the rate constant for the conversion of this complex to the final one was 3.9 s(-1); the corresponding values for PN1 were 3.4 mu M and 6.0 s(-1). By using slow-binding kinetics, it was possible to obtain estimates of the second-order rate constants for the formation of the stable thrombin-serpin complexes (1.2 x 10(4) and 1.5 x 10(6) M(-1) s(-1) for antithrombin and protease nexin 1, respectively) and the dissociation constants for these complexes (<1 nM for both serpins). The influence of viscosity on the reactions indicated that the rate of interaction of both serpins with thrombin was diffusion-controlled. Moreover, the results indicated that the initial complex reacted more rapidly to form the stable complex than it dissociated to free enzyme and inhibitor; i.e., the behavior of the serpins was analogous to that of ''sticky'' substrates. By using the results from slow-binding, viscosity, and rapid kinetic studies, it was possible to set values for all of the rate constants for the interactions of antithrombin and protease nexin 1 with thrombin. The effect of pH on the reactions indicated an essential role for the catalytic histidine of thrombin in the formation of complexes with the serpins; complexes were able to form only when this residue was in its active (unprotonated) form.