Converting trypsin to chymotrypsin: Structural determinants of S1' specificity

Trypsin and chymotrypsin differ strikingly in substrate specificities despite great similarity in their primary and tertiary structures. This work analyzes the role of two surface loops, loop 40 and loop 60, as structural determinants of the specificity of the S1'-subsite in chymotrypsin and trypsin. Chymotrypsin prefers P1' Arg/Lys residues, while trypsin prefers hydrophobic P1' residues. We replaced loop 40 and loop 60 in trypsin with their chymotrypsin counterparts. These mutations do not affect the S1 specificity and catalytic activity of trypsin. The S1' specificity was analyzed by monitoring acyl-transfer reactions to 16 amino acid amides. The exchange of loop 40 does not affect the S1' specificity. In contrast, the replacement of loop 60 causes a loss of specificity for P1'-Met/Ile/Leu. Combining both mutations reconstitutes a chymotrypsin-like S1' specificity. The specificity for Arg-NH2 increases 3-fold while the preferences for Met-NH2 and Ile-NH2 decrease 4- and S-fold, respectively. Therefore, P1'-Arg/Met discrimination changes by factor 12 and P1'-Arg/Ile discrimination changes by factor 24, Thus, loop 40 and loop Ga act synergistically to determine S1' specificity in trypsin and chymotrypsin.