Aims: Proteinase K is an extracellular endopeptidase, which is secreted by Tritirachium album Limber and belongs to the serine endopeptidase class. This enzyme is extensively applied to protein-related studies. The present study aimed at evaluating the effect of urea, guanidine hydrochloride (GnHCl), and organic solvents on the kinetic activity of proteinase K enzyme.
Materials and Methods: In this experimental study, kinetics studies were performed, using UV-Vis spectrophotometer on different concentrations of substrate, urea, and GnHCl at 40˚C and pH 7.4.
Findings: Urea decreased the V_max and K_m of enzyme at 1 and 2molar concentrations, but at higher concentrations such as 3 and 4molar, it increased enzyme activity. GnHCl had an inhibitory effect on the enzyme activity, resulting in a decrease in V_max and K_m in 1, 2, and 3molar concentrations and acted as an uncompetitive inhibitor. Organic solvents including methanol, ethanol, and isopropanol had activatory effect at low concentrations and inhibitory effect at high concentrations on the kinetic activity of proteinase K enzyme.
Conclusion: Urea has an inhibitory effect at low concentrations and an activatory effect on the activity of the enzyme at a concentrations above 2molar, but GnHCl has an inhibitory effect at all concentrations and can be used as an enzyme inhibitor. The effect of organic solvents including methanol, ethanol, and isopropanol on the activity of the proteinase K enzyme depends on their volume/volume percent; they cause enzyme activation at low percentages, but have inhibitory effect at high percentages, so that activates methanol below 30%  and isopropanol below 50%.