KINETIC MECHANISM FOR THE INTERACTION OF HIRULOG WITH THROMBIN

Hirulog (D-FPRPGGGGDGDFEEIPEEYL) is a bivalent inhibitor of thrombin consisting of a moiety (D-FPRP) that binds to the active-site cleft and a hirudin-like C-terminal region (DGDFEEIPEEYL) that binds to the positively charged surface groove of thrombin known as the anion-binding exosite. The formation of the thrombin-Hirulog complex was studied using steady-state and rapid kinetics at 37 degrees C. The inhibition constant for Hirulog was found to be 1.9 nM. Hirulog was slowly degraded by thrombin with a k(cat) value of 0.01 s(-1). The formation of the complex resulted in an enhancement of 44% in the intrinsic fluorescence of thrombin. The kinetics of the increase in thrombin fluorescence were described by a double-exponential decay. The dependence of the rate constant for the fast phase on the concentration of Hirulog could be described by the Michaelis-Menten equation with K-m and k(max) values of 0.75 +/- 0.12 mu M and 325 +/- 17 s(-1). The data were consistent with a mechanism in which the C-terminal region of Hirulog binds to the anion-binding exosite with a dissociation constant of 0.75 mu M in the first step, followed by two intramolecular steps with rate constants of about 300 and 30 s(-1). A C-terminal fragment of hirudin was found to compete in the first step confirming that this process corresponded to the binding of the hirudin-like C-terminus of Hirulog to the anion-binding exosite. The results of experiments using the fluorescent probe p-aminobenzamidine suggested that an additional very rapid step, in which Hirulog is bound to the active site, occurs between the processes with rate constants of 300 s(-1) and 30 s(-1). The steps following exosite binding resulted in 400-fold increase in the stability of the complex. The effective association rate constant at low Hirulog concentrations was 4 x 10(8) M(-1) s(-1), while the effective dissociation rate constant was about 1 s(-1); thus, under the conditions used, the release of cleaved Hirulog was not a significant pathway for the dissociation of the complex.