Attempts to convert chymotrypsin to trypsin
| Autor: | I, Venekei, L, Szilágyi, L, Gráf, W J, Rutter |
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| Rok vydání: | 1996 |
| Předmět: |
Models
Molecular Binding Sites Base Sequence Protein Conformation Molecular Sequence Data Hydrogen Bonding Saccharomyces cerevisiae Protein Structure Secondary Recombinant Proteins Chymotrypsinogen Rats Substrate Specificity Enteropeptidase Kinetics Oligodeoxyribonucleotides Mutagenesis Site-Directed Point Mutation Animals Chymotrypsin Trypsin Amino Acid Sequence Cloning Molecular Sequence Alignment |
| Zdroj: | FEBS letters. 383(1-2) |
| ISSN: | 0014-5793 |
| Popis: | Trypsin and chymotrypsin have specificity pockets of essentially the same geometry, yet trypsin is specific for basic while chymotrypsin for bulky hydrophobic residues at the P1 site of the substrate. A model by Steitz, Henderson and Blow suggested the presence of a negative charge at site 189 as the major specificity determinant: Asp189 results in tryptic, while the lack of it chymotryptic specificity. However, recent mutagenesis studies have shown that a successful conversion of the specificity of trypsin to that of chymotrypsin requires the substitution of amino acids at sites 138, 172 and at thirteen other positions in two surface loops, that do not directly contact the substrate. For further testing the significance of these sites in substrate discrimination in trypsin and chymotrypsin, we tried to change the chymotrypsin specificity to trypsin-like specificity by introducing reverse substitutions in rat chymotrypsin. We report here that the specificity conversion is poor: the Ser189Asp mutation reduced the activity but the specificity remained chymotrypsin-like; on further substitutions the activity decreased further on both tryptic and chymotryptic substrates and the specificity was lost or became slightly trypsin-like. Our results indicate that in addition to structural elements already studied, further (chymotrypsin) specific sites have to be mutated to accomplish a chymotrypsin --trypsin specificity conversion. |
| Databáze: | OpenAIRE |
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