ATTEMPTS TO CONVERT CHYMOTRYPSIN TO TRYPSIN (VOL 379, PG 143, 1996)

Trypsin and chymotrypsin have specificity pockets of essentially the s ame geometry, yet trypsin is specific for basic while chymotrypsin for bulky hydrophobic residues at the Fl site of the substrate. A model b y Steitz, Henderson and Blow suggested the presence of a negative char ge at site 189 as the major specificity determinant: Asp189 results in tryptic, while the lack of it chymotryptic specificity. However, rece nt mutagenesis studies have shown that a successful conversion of the specificity of trypsin to that of chymotrypsin requires the substituti on of amino acids at sites 138, 172 and at thirteen other positions in two surface loops, that do not directly contact the substrate. For fu rther testing the significance of these sites in substrate discriminat ion in trypsin and chymotrypsin, we tried to change the chymotrypsin s pecificity to trypsin-like specificity by introducing reverse substitu tions in rat chymotrypsin. We report here that the specificity convers ion is poor: the Ser189Asp mutation reduced the activity but the speci ficity remained chymotrypsin-like; on further substitutions the activi ty decreased further on both tryptic and chymotryptic substrates and t he specificity was lost or became slightly trypsin-like. Our results i ndicate that in addition to structural elements already studied, furth er (chymotrypsin) specific sites have to be mutated to accomplish a ch ymotrypsin --> trypsin specificity conversion.