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.