Mechanism of the inhibition of alpha-thrombin by hirudin-derived fragments hirudin(1-47) and hirudin(45-65).
The kinetic mechanism of the inhibition of alpha-thrombin by hirudin was analyzed using the hirudin-derived fragments hirudin(1-47) and hirudin(45-65). Previously, these fragments have been shown to interact with alpha-thrombin at distinct sites inhibiting thrombin-mediated clot formation. Binding to the active site the N-terminal fragment hirudin(1-47) competitively inhibits hydrolysis of the substrates Tos-Gly-Pro-Arg-NH-Mec (Tos, tosyl; NH-Mec, 4-methylcoumaryl-7-amide) and fibrinogen with Ki values of 420 +/- 18 nM and 460 +/- 25 nM, respectively. Interacting with the anion-binding site of alpha-thrombin the C-terminal fragment competitively inhibits the hydrolysis of fibrinogen with a Ki of 760 +/- 40 nM. It was found, however, that this fragment acts as a hyperbolic uncompetitive inhibitor with respect to the hydrolysis of the peptide-NH-Mec substrate. According to the Botts-Morales scheme for enzyme inhibition, the parameters Ki = 710 +/- 38 nM, K'i = 348 +/- 22 nM, as well as alpha = beta = 0.49 of thrombin inhibition by the C-terminal fragment hirudin(45-65), were obtained. The results are discussed in terms of the interaction of hirudin and thrombin.
Schmitz
T
T
Institut für Biochemie der Technischen Hochschule, Darmstadt, Federal Republic of Germany.
Rothe
M
M
Dodt
J
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry; D005340:Fibrinogen / Q000378:metabolism; D005344:Fibrinopeptide A / Q000302:isolation & purification; D005345:Fibrinopeptide B / Q000302:isolation & purification; D006629:Hirudins / Q000494:pharmacology*; D007700:Kinetics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010446:Peptide Fragments / Q000494:pharmacology*; D013379:Substrate Specificity; D013917:Thrombin / Q000037:antagonists & inhibitors*
D006629:Hirudins; D009842:Oligopeptides; D010446:Peptide Fragments; C067166:hirudin (1-47); C064473:hirudin (45-64); D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; D005340:Fibrinogen; D013917:Thrombin