pH effects in plasmin-catalysed hydrolysis of alpha-N-benzoyl-L-arginine compounds.
The steady-state kinetics of plasmin (EC 3.4.21.7) catalysed reactions with some alpha-N-benzoyl-L-arginine compounds is investigated in the pH range 5.8--9.0. The results are interpreted in terms of a three-step mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. Alpha-N-Benzoyl-L-arginine methyl ester and ethyl ester show the same pH behaviour. The kinetic parameter kc/Km is influenced by two groups with pK values of 6.5 and 8.4, respectively. kc is affected only by the group with pK equal to 6.5 and Km only by the group with pK equal to 8.4. It is suggested that the group with pK equal to 6.5 is the 1-chloro-3-tosyl-amido-7-amino-2-heptanone-sensitive histidine residue in the active site and that the group with pK equal to 8.4 is perhaps the alpha-amino group of the N-terminus in analogy to trypsin and chymotrypsin. alpha-N-Benzoyl-L-arginine amide is not hydrolysed by plasmin, but proves to be a competitive inhibitor, Ki = 12.8 +/- 1.8 mM, pH = 7.8. Also the product alpha-N-benzoyl-L-arginine is a competitive inhibitor, Ki = 26 +/- 3.1 mM, pH = 7.8. Estimates of individual rate constants are compared with similar trypsin data.
D001120:Arginine; D001565:Benzoates; D001587:Benzoylarginine-2-Naphthylamide; D001665:Binding Sites; D005341:Fibrinolysin / Q000378:metabolism*; D006639:Histidine / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D013329:Structure-Activity Relationship
D001565:Benzoates; D001587:Benzoylarginine-2-Naphthylamide; D006639:Histidine; D001120:Arginine; D005341:Fibrinolysin