The allosteric effect of salt on human mast cell tryptase.
The inhibitory effect of potassium chloride and ammonium sulphate on purified human skin tryptase and bovine trypsin was studied enzyme-kinetically, using Z-Gly-Pro-Arg-pNA, Z-Gly-Pro-Arg-AMC, benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME) as substrates. With increasing salt concentrations, the curve of reaction velocity vs. substrate concentration changed from hyperbolic to sigmoidal when anilide substrates (Z-Gly-Pro-Arg-pNA or -AMC) were used. Only the Km value increased, while the Vmax value remained unchanged. The trend was similar with BAEE or TAME as the substrates. However, the effect of salt on the hydrolysis of these ester substrates was not as strong as on the hydrolysis of anilide substrates, and sigmoidal kinetics were not observed even at the highest KCl concentration (0.7 M) used. Heparin, used as a stabilizer, had no influence on this phenomenon, but it did slightly decrease the apparent Km and Vmax values in low-salt conditions. By comparison, trypsin was not as strongly affected by salt as tryptase, and the inhibition type was mixed competitive and non-competitive. The present results indicate that the salt acts on tryptase as an allosteric effector, and this should be carefully considered when enzyme kinetic parameters and enzyme activity of skin tryptase are measured.
D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D002417:Cattle; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008407:Mast Cells / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D012867:Skin / Q000201:enzymology; D014106:Tosylarginine Methyl Ester / Q000378:metabolism
D011189:Potassium Chloride; D006493:Heparin; D014106:Tosylarginine Methyl Ester; D001120:Arginine; C009727:benzoylarginine ethyl ester; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase; D000645:Ammonium Sulfate