Structural studies of staphylococcal protease. I. Spin labelling of the active site and a comparison with other proteases.
Staphylococcus aureus protease has been spin-labelled at the active-site serine residue with the monocyclic-phosphorus spin label (MSL), 1-oxyl-2,2,6,6-tetramethyl-4-peperi-dinylethylphosphorofluoridate. The electron paramagnetic resonance (E.P.R.) sbectra of the protease in different buffers at various pH's have been analyzed and compared with those of trypsin, subtilisin BPN', and alpha-chymotrypsin under identical conditions. In a given buffer, the shape of E.P.R. signals of spin-labelled staphylococcal protease is unaffected by pH changes except below pH 4.0, at which a gradual loss of conformational integrity of the active site occurs. In bicarbonate buffer and particularly in acetate buffer, the mobility of the label is much more restricted than in phosphate buffer or in potassium chloride solution. The implications of this finding are discussed in terms of a model whereby the label is able to orient towards two different but adjacent regions of the active site. The relative population of the label in each of these orientations is believed to be buffer-dependent. An attempt to correlate the shape of the te.p.r. signals with the pH values of maximal proteolytic avtivity of the enzyme is also presented. These results show that to obtain meaningful information from a comparative spin label study of the geometry of the active site of serine proteases, particular care should be exercised to assure that the different proteases experience identical conditions of pH, buffer, and temperature.
D001665:Binding Sites; D002918:Chymotrypsin; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D010447:Peptide Hydrolases / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels; D013210:Staphylococcus / Q000201:enzymology*; D013381:Subtilisins; D014357:Trypsin
D013113:Spin Labels; D010447:Peptide Hydrolases; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin