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Assignment of histidine resonances in the sup 1 H NMR (500 MHz) spectrum of subtilisin BPN prime using site-directed mutagenesis

Journal Article · · Biochemistry; (USA)
DOI:https://doi.org/10.1021/bi00419a033· OSTI ID:6976600
;  [1]
  1. Imperial College of Science and Technology, London (England)
+ Show Author Affiliations
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{prime}. 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 pK{sub a}'s for the six histidines in this enzyme. The pK{sub a}'s of the histidine residues in subtilisin BPN{prime} 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 pK{sub a}'s in the two enzymes. It therefore appears that the assignments made for these residues in subtilisin BPN{prime} can be transferred to subtilisin Carlsberg. On the basis of these assignments, the one histidine that titrates with a substantially different pK{sub a} in the two enzymes can be assigned to histidine-238. This difference in pK{sub a} has been attributed to a Trp to Lys substitution at position 241 in subtilisin Carlsberg.
OSTI ID:
6976600
Journal Information:
Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 27:19; ISSN 0006-2960; ISSN BICHA
Country of Publication:
United States
Language:
English

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Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACIDS
AROMATICS
AZAARENES
AZOLES
BACILLUS
BACILLUS SUBTILIS
BACTERIA
BARYONS
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
HADRONS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HISTIDINE
HYDROLASES
IMIDAZOLES
INDOLES
KINETICS
LYSINE
MAGNETIC RESONANCE
MICROORGANISMS
MUTAGENESIS
MUTANTS
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PEPTIDE HYDROLASES
PH VALUE
PROTONS
PYRROLES
REACTION KINETICS
RESONANCE
SERINE PROTEINASES
SPECTRA
TRYPTOPHAN