The differential specificity of chymotrypsin A and B is determined by amino acid 226

The A and B isoforms of the pancreatic serine proteinase, chymotrypsin are known to cleave substrates selectively at peptide bonds formed by some hydr ophobic residues, like tryptophan, phenylalanine and tyrosine. We found, ho wever, that the B forms of native bovine and recombinant rat chymotrypsins are two orders of magnitude less active on the tryptophanyl than on the phe nylalanyl or tyrosyl substrates, while bovine chymotrypsin A cleaves all th ese substrates with comparable catalytic efficiency. Analysing the structur e of substrate binding pocket of chymotrypsin A prompted us to perform an A la226Gly substitution in rat chymotrypsin B. The specificity profile of the Ala226Gly rat chymotrypsin B became similar to that of bovine chymotrypsin A suggesting that only the amino acid at sequence position 226 is responsi ble for the differential specificities of chymotrypsin A and B isoenzymes.