Catalysis by trypsin

Abstract

Investigations are reported concerning the mechanism of catalysis by an enzyme of known structure, bovine trypsin. Evidence for proton transfers at the active site residues His 57 and Asp 102 during catalysis is provided by experimental techniques which specifically detect the ionization state of either the carboxyl group or the imidazole ring. The pK_a of Asp 102 in a chemically modified—but still active--form of trypsin is shown to be 6.8 by difference infrared spectroscopic titration. This assignment is facilitated by the use of inhibitory copper (Cu^(++)) ion which lowers the pK_(app) of Asp 102 by binding to trypsin between Asp 102 and His 57, as is demonstrated crystallographically. Information concerning the solvent accessibilities and mobilities of the three imidazole side chains of bovine trypsin is provided by measurements of the pH dependences of the rates of exchange of the ring C-2 protons with tritium in labeled water. Kinetic studies of substrate hydrolysis do not detect the pK_a of His 57 anywhere in the range 3 - 8.5. Pre-incubation of trypsin at low or at high pH shows that the failure to detect the pK_a of His 57 kinetically is not due to a slow pH-dependent conformational change. The implications of the assignment of a pK_a of 6. 8 to Asp 102, and of the pK_a of His 57 being below 3, are discussed with regard to the catalytic mechanism of the serine proteases.