Assignment of the Nε2H and Nδ1H Resonances at the Active-Center Histidine in Chymotrypsin and Subtilisin Complexed to Peptideboronic Acids without Specific 15N Labeling1

A combination of 1 H and 15 N nuclear magnetic resonance experiments have been carried out to assign the two high-frequency 1 H resonances that result from the complexation of subtilisin E and MeoSuc-Ala-Ala-Pro-boroPhe, a potent peptideboronic acid inhibitor of both subtilisins from a variety of sources and chymotrypsin. First, it was demonstrated unequivocally using two auxotrophs of Bacillus subtilis that the proton resonances at 16 and 17 ppm pertain to a histidine residue. Next it was shown by both 1D and 2D methods that the two proton resonances pertain to the same histidine . Finally, in the subtilisin E-peptideboronate complex, all of the imidazole proton and nitrogen resonances pertinent to this His64 were assigned as follows: N ε2 at 183 and N δ1 at 189 ppm; N ε2 H at 16 and N δ 1 H at 17.4 ppm; C ε1 H at 9.20 and C δ2 H at 7.09 ppm. Using the 1D NOE method demonstrated on the subtilisin−peptideboronate complex, the resonances due to complexation were also assigned in the chymotrypsin−peptideboronate complex. The assignments of the two high-frequency resonances are reversed from those assumed in a previous paper from the current authors (Zhong, S.; Haghjoo, K.; Kettner, C.; Jordan, F. J. Am. Chem. Soc . 1995, 117, 7047−7055), in which the assignments were adopted from the relative chemical shifts assigned on α-lytic protease [Bachovchin, W. W.; Wong, W. Y. L.; Farr-Jones, S.; Shenvi, A. B.; Kettner, C. A. Biochemistry 1988, 27, 7689−7697].