Designing substrate specificity by protein engineering of electrostatic interactions.

J A Wells; D B Powers; R R Bott; T P Graycar; D A Estell

doi:10.1073/pnas.84.5.1219

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Designing substrate specificity by protein engineering of electrostatic interactions.

J A Wells·D B Powers·R R Bott·T P Graycar·D A Estell

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Abstract

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.

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Binding Sites Electrochemistry Genes Genetic Engineering Models, Molecular Mutation Protein Conformation Recombinant Proteins Substrate Specificity Subtilisins X-Ray Diffraction Journal Article Research Support, Non-U.S. Gov't

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J A Wells, D B Powers, R R Bott, T P Graycar, D A Estell. 1987. Designing substrate specificity by protein engineering of electrostatic interactions.. https://doi.org/10.1073/pnas.84.5.1219

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