Ala226 to Gly and Ser189 to Asp mutations convert rat chymotrypsin B to a trypsin-like protease.
In a previous successful attempt to convert trypsin to a chymotrypsin-like protease, 15 residues of trypsin were replaced with the corresponding ones in chymotrypsin. This suggests a complex mechanism of substrate recognition instead of a relatively simple one that only involves three sites, residues 189, 216 and 226. However, both trypsin-->elastase and chymotrypsin-->trypsin conversion experiments carried out according to the complex model resulted in non-specific proteases with low catalytic activity. Chymotrypsin used in the latter studies was of B-type, containing an Ala residue at position 226. Trypsins, however, contain a conserved Gly at this site. The substantially decreased trypsin-like activity of the G226A trypsin mutant also suggests a specific role for this site in substrate binding. Here we investigate the role of site 226 by introducing the A226G substitution into chymotrypsin-->trypsin mutants which were constructed according to both the simple (S189D mutant) and the complex model (S(1) mutant) of specificity determination. The kinetic parameters show that the A226G substitution in the S(1) mutant increased the chymotrypsin-like activity, while the trypsin-like activity did not change. In contrast, this substitution in the S189D chymotrypsin mutant resulted in a 100-fold increase in trypsin-like activity and a trypsin-like specificity profile as tested on a competing oligopeptide substrate library. Additionally, the S189D+A226G mutant is the first trypsin-like chymotrypsin that undergoes autoactivation, an exclusive property of trypsinogen among pancreatic serine proteases.
Jelinek
Balázs
B
Biotechnology Research Group of the Hungarian Academy of Sciences.
Antal
József
J
Venekei
István
I
Gráf
László
L
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001550:Benzamidines / Q000494:pharmacology; D002918:Chymotrypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002919:Chymotrypsinogen / Q000378:metabolism; D005998:Glycine / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010447:Peptide Hydrolases / Q000187:drug effects / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology
D001550:Benzamidines; D014361:Trypsin Inhibitors; D001224:Aspartic Acid; D012694:Serine; D002919:Chymotrypsinogen; D010447:Peptide Hydrolases; D002918:Chymotrypsin; C117609:chymotrypsin B; D014357:Trypsin; C032157:benzamidine; D000409:Alanine; D005998:Glycine