title: Assignment of histidine resonances in the 1H NMR (500 MHz) spectrum of subtilisin BPN' using site-directed mutagenesis.
abstract: A spin-echo pulse sequence has been used to resolve the six histidine C-2H protons in the 500-MHz NMR spectrum of subtilisin BPN'. Five of these residues have been substituted by site-directed mutagenesis, and this has enabled a complete assignment of these protons to be obtained. Analysis of the pH titration curves of these signals has provided microscopic pKas for the six histidines in this enzyme. The pKas of the histidine residues in subtilisin BPN' have been compared with the values obtained for the histidines in the homologous enzyme from Bacillus licheniformis (subtilisin Carlsberg). Four of the five conserved histidines titrate with essentially identical pKa's in the two enzymes. It therefore appears that the assignments made for these residues in subtilisin BPN' can be transferred to subtilisin Carlsberg. On the basis of these assignments, the one histidine that titrates with a substantially different pKa in the two enzymes can be assigned to histidine-238. This difference in pKa has been attributed to a Trp to Lys substitution at position 241 in subtilisin Carlsberg.
authors.affiliation: Department of Chemistry, Imperial College of Science and Technology, London, U.K.
authors.forename: M
authors.initials: M
authors.lastname: Bycroft
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D003001:Cloning, Molecular; D005973:Glutamine; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D013381:Subtilisins / Q000235:genetics
chemical_list: D005973:Glutamine; D006639:Histidine; D013381:Subtilisins