Proton inventory studies, kinetic studies in mixtures of protium oxide and deuterium oxide, will be used in studies of the mechanism of action of the enzyme Alpha-chymotrypsin. A model cationic peptide surfactant which has been suggested to mimic the """"""""charge-relay"""""""" chain of Alpha-chymotrypsin will be studied as well. The primary aims of the research relate to the so-called """"""""charge-relay"""""""" mechanism of serine proteases. Alpha-Chymotrypsin modified by methylation at N-3 of histidine-57, a key component of the active site, will be studied in both acylation and deacylation reactions to outline the role of the """"""""charge-relay"""""""" mechanism in catalysis by serine proteases. The origin of the large solvent isotope effects in a system where the classic charge-relay mechanism is precluded will provide mechanistic details not available from any other studies. A highly attractive """"""""charge-relay"""""""" model system will also be mechanistically defined using the technique. In essence, the proposed research will allow us to determine whether or not previously determined proton inventories for Alpha-chymotrypsin reactions which have been interpreted in terms of a charge-relay mechanism are valid or not. Kossiakoff and Spencer (1) have concluded, based on neutron diffraction studies, that the interpretation is incorrect.
