title: Free energy perturbation calculations on binding and catalysis after mutating threonine 220 in subtilisin.
abstract: We present the results of free energy perturbation calculations on binding and catalysis of a tetrapeptide substrate, acetyl-Phe-Ala-Ala-Phe-NMe, by native subtilisin BPN' and a subtilisin BPN' mutant (Thr220----Ala220). The calculated difference in the free energy of binding was 0.70 +/- 0.72 kcal/mol. The calculated difference in the free energy of catalysis was 1.48 +/- 0.89 kcal/mol. These calculated values compare well with the experimental values in which another substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, was used. These findings suggest that Thr220 is more important for catalysis than substrate binding.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
authors.forename: N
authors.initials: N
authors.lastname: Mizushima
authors.forename: D
authors.initials: D
authors.lastname: Spellmeyer
authors.forename: S
authors.initials: S
authors.lastname: Hirono
authors.forename: D
authors.initials: D
authors.lastname: Pearlman
authors.forename: P
authors.initials: P
authors.lastname: Kollman
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001412:Bacillus subtilis / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D013381:Subtilisins