Hydrogen bonding to active-site histidine in peptidyl boronic acid inhibitor complexes of chymotrypsin and subtilisin: Proton magnetic resonance assignments and H D fractionation

H-1 NMR chemical shift assignments were established for (NH)-H-delta 1 (16. 9 ppm) and (NH)-H-epsilon 2 (16.1 ppm) of the active-center His57 for the c omplex of MeOSuc-Ala-Ala-Pro-boroPhe (BoroPhe) with chymotrypsin and for th e (CH)-H-epsilon 1 proton (9.2 ppm at low pH and 8.5 ppm at high pH) of His 57 in uncomplexed chymotrypsin. The assignment for (CH)-H-epsilon 1 correct s previous assignments and reveals an unusual environment of this carbon-bo und proton. The relative NH assignments are reversed from the order of NH a ssignments previously found for alpha-lytic protease complexes with boronat e inhibitors. Isotopic fractionation factors (H/D) were determined using H- 1 NMR for hydrogen bonds to the active site histidine in BoroPhe complexes with chymotrypsin and subtilisin E, and for uncomplexed chymotrypsin. Measu red fractionation factors accurate to about +/-0.1 were 0.82 (pH 10) and 0. 64 (pH 3) for the (NH)-H-delta 1 proton of uncomplexed chymotrypsin. In the presence of BoroPhe at pH 6.5, the (NH)-H-delta 1 fractionation factors we re 0.65 for the chymotrypsin-inhibitor complex, and 0.53 for the subtilisin -inhibitor complex. Measurements for the (NH)-H-epsilon 2 fractionation fac tor were 1.05 (uncomplexed chymotrypsin at pH 10), 0.93 (BoroPhe-chymotryps in at pH 6.5), and 0.76 (BoroPhe-subtilisin at pH 6.5). Both model calculat ions of isotopic fractionation factors and experimentally determined inhibi tion constants were used in the analysis of the fractionation-factor result s.