Protein engineering of chymosin; modification of the optimum pH of enzyme catalysis
| Autor: | James E. Pitts, Dimitris Mantafounis |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
Protein Conformation
Stereochemistry Molecular Sequence Data Bioengineering Protein Engineering Biochemistry Catalysis Enzyme catalysis Scissile bond Protein structure Escherichia coli Amino Acid Sequence Chymosin Binding site Site-directed mutagenesis Molecular Biology Binding Sites biology Hydrogen bond Chemistry Active site Hydrogen Bonding Hydrogen-Ion Concentration Mutation biology.protein Biotechnology |
| Zdroj: | "Protein Engineering, Design and Selection". 3:605-609 |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/3.7.605 |
| Popis: | The aspartic proteinase chymosin exhibits a local network of hydrogen bonds involving the active site aspartates and surrounding residues which may have an influence on the rate and optimal pH of substrate cleavage. We have introduced into chymosin B the following substitutions: Asp304 to Ala (D304A), Thr218 to Ala (T218A) and Gly244 to Asp (G244D, chymosin A), using oligonucleotide-directed mutagenesis. Kinetic analysis of these active mutants shows shifts in their pH optima to 4.4 D304A, 4.2 T218A and 4.0 G244D compared with 3.8 for chymosin B using a synthetic octapeptide substrate. The upward shift of the D304A and T218A may be due to the loss of hydrogen bond interactions indirectly affecting the catalytic aspartates 32 and 215. The G244D mutation which is in a flexible loop on the surface of the enzyme may alter the conformation of the specificity pockets on the prime side of the scissile bond. |
| Databáze: | OpenAIRE |
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