| Autor: |
Wells, J A, Powers, D B, Bott, R R, Graycar, T P, Estell, D A |
| Zdroj: |
Proceedings of the National Academy of Sciences of the United States of America; March 1987, Vol. 84 Issue: 5 p1219-1223, 5p |
| Abstrakt: |
Protein engineering of electrostatic interactions between charged substrates and complementary charged amino acids, at two different sites in the substrate binding cleft of the protease subtilisin BPN', increases kcat/Km toward complementary charged substrates (up to 1900 times) and decreases kcat/Km toward similarly charged substrates. From kinetic analysis of 16 mutants of subtilisin and the wild type, the average free energies for enzyme-substrate ion-pair interactions at the two different sites are calculated to be -1.8 +/- 0.5 and -2.3 +/- 0.6 kcal/mol (1 cal = 4.18 J) [at 25 degrees C in 0.1 M Tris X HCl (pH 8.6)]. The combined electrostatic effects are roughly additive. These studies demonstrate the feasibility for rational design of charged ligand binding sites in proteins by tailoring of electrostatic interactions. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
|
