The double catalytic triad, Cys25-His159-Asp158 and Cys25-His159-Asn175, in papain catalysis: role of Asp158 and Asn175.

The 1.65 A X-ray structure of papain, which exhibits a Cys25-His159-Asn175 triad, does not correspond to the catalytically active ion pair state since Cys25 is oxidized to cysteic acid and His159 is predominantly neutral. Thus, stochastic boundary molecular dynamics simulations starting from the 1.65 A X-ray structure of papain have been performed for Cys25 and His159 in the SH-ImH+, SH-Im, S(-)-ImH+ and S(-)-Im states and for Asp158 mutated to Asn, Glu and Gly in the ion pair state. By comparing the resulting averaged structures and analyzing the trajectories of certain interatomic distances, important differences in the active-site geometry of papain have been found. In particular, the initial Cys25(S-)-His159(ImH+)-Asn175(C = O) triad found in the X-ray structure is retained in all the structures except the wild type and Asp158-->Asn ion pair states where there is a conformational transition to form the triad, Cys25(S-)-His159(ImH+)-Asp158(COO-). Both triads, Cys25(S-)-His159(ImH+)-Asp158(COO-) and Cys25(S-)-His159(ImH+)-Asn175(C = O) are postulated to participate in catalysis and their roles are discussed. Thus, catalysis does not take place from a single steric position but a two-state mechanism.