131 Biochimica et Biophysica Acta, 527 (1978) 131--141 © Elsevier/North-Holland Biomedical Press BBA 68579 CATALYSIS AND LEAVING GROUP BINDING IN ANILIDE HYDROLYSIS BY CHYMOTRYPSIN DIMITER PETKOV, EVDOKIA CHRISTOVA and IVANKA STOINEVA Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia (Bulgaria) (Received April 13th, 1978) Summary The influence of the leaving group on the reactivity of specific anilides in a-chymotrypsin-catalyzed hydrolysis (chymotrypsin, EC 3.4.21.2) involves both its binding to the enzyme (steric effect) and electronic nature (electronic effect). These effects are considered in terms of the stereoelectronic theory for the formation and cleavage of the tetrahedral intermediate in acyltrans- fer reactions. The application of this theory to the enzyme hydrolysis leads to the conclusion that the nature of the reaction products and the effec- tiveness of the catalysis are controlled by the orientation of the leaving group nitrogen lone pair orbital. The leaving group binding affects the formation of a reactive conformation of the enzyme tetrahedral intermediate that is pre- sumed to intervene between the Michaelis complex and the acylenzyme. The steric and electronic effects could be separated in a straightforward fashion only in the case of equal binding of the leaving groups to the leaving-group- binding site of a-chymotrypsin. Introduction Although a-chymotrypsin-catalyzed hydrolysis of specific anilides has been extensively studied, the results are still inconclusive. The electronic effects of the substituents in the aniline ring on the steady-state parameters are the basic mechanistic information in these studies. It is, however, difficult to separate these effects from the steric effects of the substituents. Assuming that the steric effects are small compared to the electronic effects, positive [1--3], nega- tive [4] and no [5,6] substituent dependences have been found. These con- tradictory data and the compelling evidence for the presence'of a leaving group All correspondence should be sent to: D. Petkov, Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. Abbreviation: KEF , enzyme-proflavin binding constant.