Proton magnetic resonance titration curves of the three histidine residues of staphylococcal protease.
Proton magnetic resonance spectra of staphylococcal protease, a serine protease from Staphylococcus aureus, strain V8, are presented. Initial proton spectra were obtained at 220 MHz, and more detailed studies of the aromatic region were carried out by correlation spectroscopy at 250 MHz. The overall spectrum bears a close resemblance to one calculated from the sum of spectra of the component amino acids. Chemical shifts of the three tyrosine, four phenylalanine, and three histidine residues appear to be equivalent at pH 3.7 and 8.5 indicating that they are all in normal chemical environments in the enzyme. The staphylococcal protease contains a large number of slowly exchanging protons. In fact, interpretable spectra of the aromatic region were obtained only after extensive exchange of N-H groups with deuterium from the D2O solvent. Proton magnetic resonance titration studies of the three histidine residues indicate that these have normal chemical shifts and pK' values. When the data are fitted to single noninteracting titration curves, the histidine pK' values are 7.19 plus or minus 0.02, 6.85 plus or minus 0.03, and 6.69 plus or minus 0.02. The titration curves of two of the histidine residues indicate negative cooperativity. A possible explanation for this is a direct electrostatic interaction between the two histidines. The titration data for these histidines give a significantly better fit to such a mutual interaction model than to noninteracting titration curves. The component microscopic dissociation constants have been calculated. Mutual interaction leads to pK' displacements of 0.31 unit; which indicates a distance of approximately 7 angstrom between the two interacting histidine rings according to the model of Tanford and Roxby. The proton resonances of the two interacting histidines are doubled in the pH region 6.7-7.0 suggesting the presence of two forms of the enzyme having lifetimes in excess of 30 msec.
D000596:Amino Acids / Q000032:analysis; D006639:Histidine / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases; D010649:Phenylalanine / Q000032:analysis; D011487:Protein Conformation; D012694:Serine; D013210:Staphylococcus / Q000201:enzymology*; D014443:Tyrosine / Q000032:analysis
D000596:Amino Acids; D010446:Peptide Fragments; D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; D006639:Histidine; D010447:Peptide Hydrolases