Recombinant hirudin: kinetic mechanism for the inhibition of human thrombin.
Recombinant hirudin variant-2(Lys47), was found to be a competitive inhibitor of human alpha-thrombin with respect to peptidyl p-nitroanilide substrates. These results contrast with those of Degryse and coworkers that suggest that recombinant hirudin variant-2(Lys47) inhibited thrombin by a noncompetitive mechanism [Degryse et al. (1989) Protein Engng, 2, 459-465]. gamma-Thrombin, which can arise from alpha-thrombin by autolysis, was shown to have an affinity for recombinant hirudin variant-2(Lys47) that was four orders of magnitude lower than that of alpha-thrombin. It was demonstrated that the apparent noncompetitive mechanism observed previously was probably caused by a contamination of the thrombin preparation by gamma-thrombin. Comparison of the inhibition of alpha-thrombin by recombinant hirudins variant-2(Lys47) and variant-1, which differ from one another in eight out of 65 amino acids, indicated that the two variants have essentially the same kinetic parameters.
Stone
S R
SR
Friedrich Miescher-Institut, Basel, Switzerland.
Hofsteenge
J
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D006629:Hirudins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000502:physiology
D006629:Hirudins; D011994:Recombinant Proteins; D013917:Thrombin