1H NMR chemical shift assignments were established for Nδ1H (16.9 ppm) and Nε2H (16.1 ppm) of the active-center His57 for the complex of MeOSuc-Ala-Ala-Pro-boroPhe (BoroPhe) with chymotrypsin and for the Cε1H proton (9.2 ppm at low pH and 8.5 ppm at high pH) of His57 in uncomplexed chymotrypsin. The assignment for Cε1H corrects previous assignments and reveals an unusual environment of this carbon-bound proton. The relative NH assignments are reversed from the order of NH assignments previously found for α-lytic protease complexes with boronate inhibitors. Isotopic fractionation factors (H/D) were determined using 1H NMR for hydrogen bonds to the active site histidine in BoroPhe complexes with chymotrypsin and subtilisin E, and for uncomplexed chymotrypsin. Measured fractionation factors accurate to about ±0.1 were 0.82 (pH 10) and 0.64 (pH 3) for the Nδ1H proton of uncomplexed chymotrypsin. In the presence of BoroPhe at pH 6.5, the Nδ1H fractionation factors were 0.65 for the chymotrypsin−inhibitor complex, and 0.53 for the subtilisin−inhibitor complex. Measurements for the Nε2H fractionation factor were 1.05 (uncomplexed chymotrypsin at pH 10), 0.93 (BoroPhe−chymotrypsin at pH 6.5), and 0.76 (BoroPhe−subtilisin at pH 6.5). Both model calculations of isotopic fractionation factors and experimentally determined inhibition constants were used in the analysis of the fractionation-factor results.

A combination of 1H and 15N nuclear magnetic resonance experiments have been carried out to assign the two high-frequency 1H 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:  Nε2 at 183 and Nδ1 at 189 ppm; Nε2H at 16 and Nδ1H at 17.4 ppm; Cε1H at 9.20 and Cδ2H 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].