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. Thi s work analyzes the role of two surface loops, loop 40 and loop 60, as structural determinants of the specificity of the S1'-subsite in chym otrypsin and trypsin. Chymotrypsin prefers P1' Arg/Lys residues, while trypsin prefers hydrophobic P1' residues. We replaced loop 40 and loo p 60 in trypsin with their chymotrypsin counterparts. These mutations do not affect the S1 specificity and catalytic activity of trypsin. Th e S1' specificity was analyzed by monitoring acyl-transfer reactions t o 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 o f specificity for P1'-Met/Ile/Leu. Combining both mutations reconstitu tes a chymotrypsin-like S1' specificity. The specificity for Arg-NH2 i ncreases 3-fold while the preferences for Met-NH2 and Ile-NH2 decrease 4- and S-fold, respectively. Therefore, P1'-Arg/Met discrimination ch anges by factor 12 and P1'-Arg/Ile discrimination changes by factor 24 , Thus, loop 40 and loop Ga act synergistically to determine S1' speci ficity in trypsin and chymotrypsin.