Hydrogen Bonding to Active-Site Histidine in Peptidyl Boronic Acid Inhibitor Complexes of Chymotrypsin and Subtilisin: Proton Magnetic Resonance Assignments and H/D Fractionation

1H NMR chemical shift assignments were established for Nδ1H (16.9 ppm) and Ne2H (16.1 ppm) of the active-center His57 for the complex of MeOSuc-Ala-Ala-Pro-boroPhe (BoroPhe) with chymotrypsin and for the Ce1H proton (9.2 ppm at low pH and 8.5 ppm at high pH) of His57 in uncomplexed chymotrypsin. The assignment for Ce1H 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 compl...