Direct proton magnetic resonance determination of the pKa of the active center histidine in thiolsubtilisin

The serine proteases constitute a group of endopeptidases whose members owe their catalytic activity to the presence of a catalytic triad of amino acids consisting of a serine, a histidine and an aspartate. The pKa values for this histidine have been determined for several cases in which there is a negative charge installed at the serine to mimic the oxyanionic intermediate and related transition state for the catalytic pathway. Instances from this laboratory include (1) replacement of the serine by a cysteine in subtilisin to create a thiolate; (2) formation of monoisopropylphosphoryl‐Ser 195 monoanionic phosphodiesters (in trypsin and chymotrypsin, Ser 221 in subtilisins); and (3) tetrahedral boronates formed with peptide boronic acids. The nuclear magnetic resonance (NMR) signals pertinent to this histidine, or signals indirectly reflecting the state of ionization of this histidine, have been used effectively to monitor changes in the active center ionization state. In every case studied, there is elevation of the pKa at the histidine when the negative charge is installed at the serine position. Herein is reported the first NMR measurement of the active center His 63 pKa in thiolsubtilisin Carlsberg; it is elevated by 3 units compared with the parent enzyme. Using a numerical solution (finite difference) of the Poisson‐Boltzmann equation, a protein dielectric constant of 4 provides a good estimate of the experimentally observed pKa elevations. Very significantly, a very low protein dielectric constant (εp = 3–5) is required in all of the comparisons, and for all three enzymes used (chymotrypsin, trypsin, and subtilisin). Finally, we discuss why the electrostatic perturbation sensed at His of the active center is more amplified by a negative charge on the Ser side than the same charge on the Asp side. A plausible explanation is that the positive charge on the imidazolium ring of the His is localized, with the Nδ1 carrying a smaller fraction, the Nε2 carrying the bulk of the positive charge.