A PROTEIN ENGINEERING STUDY OF THE ROLE OF ASPARTATE-158 IN THE CATALYTIC MECHANISM OF PAPAIN

The controversy concerning the various suggested roles for the side chain of Aspl58 in the active site of papain has been clarified by using site-directed mutagenesis. Both wild-type papain and an Asp l58Asn variant were produced in a baculovirus-insect cell expression system, purified to homogeneity from the culture, and characterized kinetically. With CBZ-Phe-Arg-MCA as substrate, the kcat/KM and kcat values obtained for the Aspl58Asn papain are 20000 M−1•s−1 and 34 s−1 respectively, as compared with values of 120000 M−1•s−1 and 51 s−1 obtained for the wild-type papain. In addition, the pH-(kcat/KM) profile for the Aspl58Asn enzyme is shifted relative to that for the wild-type enzyme to lower values by approximately 0.3 pH unit. This shows clearly that Asp 158 is not, as previously postulated, an essential catalytic residue. In addition, the pH dependency data are interpreted to indicate that, contrary to earlier suggestions, the negatively charged side chain of Aspl58 does not significantly stabilize the active-site thiolate-imidazolium ion pair. However, its presence does influence the pKa’s associated with ion-pair formation in a manner compatible with electrostatic considerations. © 1990, American Chemical Society. All rights reserved.