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 N(δ1)H (16.9 ppm) and N(ε2)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(ε1)H proton (9.2 ppm at low pH and 8.5 ppm at high pH) of His57 in uncomplexed chymoptrypsin. The assignment for C(ε1)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 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 N(δ1)H proton of uncomplexed chymotrypsin. In the presence of BoroPhe at pH. 6.5, the N(δ1)H fractionation factors were 0.65 for the chymotrypsin- inhibitor complex, and 0.53 for the subtilisin-inhibitor complex. Measurements for the N(ε2)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.