Evolutionary Divergence of Substrate Specificity within the Chymotrypsin-like Serine Protease Fold

We have described a structural scaffold extant in over 500 homologous enzymes, where the catalytic mechanism is identical and the substrate specificity varied. The many known structures of chymotrypsin-fold serine proteases delineate a clear framework showing that this variability is a function of evolved diversity in the structures of surface loops, which surround the extended substrate binding site. Because the loops are conserved in their relative positions with respect to the N to C direction of the scissile peptide, general themes for their individual functions can be described. Thus, loop C is invariably positioned to directly contact the extended substrate on the N-terminal side of the scissile bond, whereas loops A, B, D, and E interact on the leaving group side (Fig. 3). Loops 2 and 3 can modulate either or both of the S1 sites or the subsite preferences on the N-terminal side of the scissile bond. Loop 1 is thus far known to determine only the S1 site specificity.