title: Designing substrate specificity by protein engineering of electrostatic interactions.
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.
mesh_terms: D001665:Binding Sites; D004563:Electrochemistry; D005796:Genes; D005818:Genetic Engineering / Q000379:methods*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D011994:Recombinant Proteins; D013381:Subtilisins