The Mechanism of Trypsin Catalysis at Low pH PROPOSAL FOR

Evidence is presented for the formation of E = S being a pseudo-equilibrium process with a minimum value for the rate constant for the formation of E S complex of -2 X 10’ M“ S-* . The pH dependences have been determined for the dissociation constant of the E S complex (Kb), as well as for the individual rate constants associated with the catalytic steps (k+2 and k+3). The pH profile of k+a/K,(= kCat/Km) reflects the ionization of groups of pK, value 4.5 +0.1, and 7.1 k 0.1 in the free enzyme (T = 21 k 1 “C, Z = 0.1). The pH dependence of k+2 implicates two ionizing groups in the acylation step with p& 3.75 f 0.1 and 7.1 0.1. The change in the value of K. with pH indicates a change in pK, of a group on binding the substrate. The pH dependence of k+3 implicates only one ionizing group in the catalytic step of pK, 7.1 f 0.1. The pH dependence of the kinetic parameters shows that at acid pH values (2.55-4.80), the k,, step is ratelimiting in catalysis, whereas for pH values higher than 4.80, k+z becomes rate-limiting. The change in K. and in the rate-limiting step with pH may be related to the acid-base equilibrium of Asp 177 present in the recognition site of trypsin. Steady state and pre-steady state parameters for the trypsin-catalyzed hydrolysis of the noncationic substrate Q benzyloxycarbonyl L alanine p nitrophenyl ester (ZANPE) in the presence and absence of ethylammonium ion have been studied in the pH range between 3.0 and 8.0. In the absence of ethylammonium ion the pH dependence of kinetic parameters for the trypsincatalyzed hydrolysis of ZANPE shows that the k+z step is rate-limiting in catalysis over the whole pH range explored. The presence of ethylammonium ion induced changes in the rate-limiting step with pH similar to those reported for ZLNPE. In addition, the affinity constant for the reaction of ethylammonium ion with trypsin changes with pH, and implies a pK, shift from 4.55 k 0.1 in the free nzyme to 3.75 2 0.1 in the enzyme-