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 kcat 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 Km and kmax values of 0.75 +/- 0.12 microM 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 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Parry
M A
MA
Department of Haematology, University of Cambridge, U.K.
Maraganore
J M
JM
Stone
S R
SR
D000595:Amino Acid Sequence; D001550:Benzamidines / Q000494:pharmacology; D005470:Fluorometry; D006629:Hirudins / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D001550:Benzamidines; D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C006500:4-aminobenzamidine; D013917:Thrombin; C074619:bivalirudin