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.