Histidyl and tyrosyl residue ionization studies of subtilisin Novo

The low field portion of the 360 MHz 1H nuclear magnetic resonance spectrum of phenylmethanesulfonyl-subtilisin Novo (EC 3.4.21.14) has been studied as a function of pH. Analysis of the pH-induced chemical shift changes occurring between 6 to 7 ppm revealed five classes of ionizable residues with pK values (uncorrected) of 10.3, 10.7, 10.7, 10.8, and 11.0. With a single exception the titration curves can be fit by assuming a simple proton ionization equilibrium. Four classes of low intensity broad resonances, assigned to the histidyl residues, are observed between 8 and 9 ppm. Uncorrected pK values of 5.4, 5.7, 6.0, and 6.4 were determined for the residues comprising each of these classes. The spectral data are consistent with protonation of one or more histidyl residues upon acid induced denaturation of the protein. These results are compared with those of analogues studies performed by the use of other techniques.