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

1 H NMR chemical shift assignments were established for N δ 1 H (16.9 ppm) and N ε 2 H (16.1 ppm) of the active-center His57 for the complex of MeOSuc-Ala-Ala-Pro-boroPhe (BoroPhe) with chymotrypsin and for the C ε 1 H proton (9.2 ppm at low pH and 8.5 ppm at high pH) of His57 in uncomplexed chymotrypsin. The assignment for C ε 1 H 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 1 H 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 N δ 1 H proton of uncomplexed chymotrypsin. In the presence of BoroPhe at pH 6.5, the N δ 1 H fractionation factors were 0.65 for the chymotrypsin−inhibitor complex, and 0.53 for the subtilisin−inhibitor complex. Measurements for the N ε 2 H 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.