Assignment of the Nε2H and Nδ1H resonances at the active-center histidine in chymotrypsin and subtilisin complexed to peptideboronic acids without specific 15N labeling

A combination of H-1 and N-15 nuclear magnetic resonance experiments have been carried out to assign the two high-frequency H-1 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 mio 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-epsilon 2 at 183 and N-delta 1 at 189 ppm; (NH)-H-epsilon 2 at 16 and (NH)-H-delta 1 at 17.4 ppm: (CH)-H-epsilon 1 at 9.20 and (CH)-H-delta 2 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 alpha-lytic protease [Bachovchin, W. W.; Wong, W. Y. L.; Farr-Jones, S.; Shenvi, A. B.; Kettner, C. A. Biochemistry 1988, 27, 7689-7697].