Rate-determining steps in penicillopepsin-catalysed reactions

Abstract The hydrolysis of Ac-(Ala) 2 -Lys-Nph-(Ala) 2 -amide ( II ) by penicillopepsin is characterized by a solvent isotope effect of 2.11, whereas the hydrolysis of Ac-Lys-Nph-amide ( I ) shows no solvent isotope effect. The dependence of the isotope effect on the concentration of D 2 O in H 2 O for substrate II is not linear and suggests that two or more protons are involved in its rate-determining step. We propose that for substrate I the rate-determining step is the distortion of the scissile bond towards a tetrahedral configuration, and for substrate II a conformational change induced by the occupation of the S 3 pocket in the enzyme.