Role of serine 195 in the stabilization of β-trypsin and β-trypsin-ligand complexes

Abstract

β-Anhydrotrypsin, an inactive derivative of trypsin obtained by specific dehydration of Ser 195 of the enzyme active site, is more stable than the native enzyme to the denaturing effect of guanidine·HCl between pH 3 and pH 10. Calcium ion binds with the same affinity to β-anhydrotrypsin and to β-trypsin, whereas benzamidine binds to the modified enzyme with a dissociation equilibrium constant four time higher than that found for β-trypsin. However, both ligands are still able to protect β-anhydrotrypsin agianst the action of denaturing agents. From the protective effect induced by the proteinase inhibitors on β-trypsin and β-anhydrotrypsin against the action of secondary bond breaking agents, two categories of inhibitors may be distinguished. (1) Those which have a Lys residue in their reactive site: pancreatic trypsin inhibitor (Kunitz) and lima bean inhibitor. They are strong inhibitors of β-trypsin and, when bound to the enzyme, they stabilize its structure against denaturation and transconformation in the acid and alkaline region in 3·5 m -guanidine·HCl. Pancreatic trypsin inhibitor (Kunitz) has a similar protective effect on β-anhydrotrypsin structure, but in the case of lima bean inhibitor, no protective effect is observed on β-anhydrotrypsin under pH 7. (2) The arginyl inhibitors: soybean trypsin inhibitor (Kunitz) and ovomucoid trypsin inhibitor. They are weaker inhibitors of β-trypsin than the lysyl ones and do not protect the enzyme against its acidic transconformation in 3·5 m -guanidine·HCl. These inhibitors do not protect β-anhydrotrypsin either in the acidic or in the alkaline pH range. From the above results, the following conclusions arise: Ser 195 is not necessary for the stabilization of the complexes formed between trypsin and pancreatic trypsin inhibitor (Kunitz) from pH 5 to pH 9, and between trypsin and lima bean inhibitor from pH 7 to pH 9 in guanidine·HCl. However, this residue is essential for the stabilization of the complexes formed between trypsin and soybean trypsin inhibitor and ovomucoid trypsin inhibitor in guanidine·HCl. These results show that if the mechanism of interaction between trypsin and its proteinase inhibitors is supposed to be unique, nevertheless Ser 195 plays a very different role in the stabilization of the various enzyme-inhibitor complexes.