Assignment of the N^ε2H and N^δ1H Resonances at the Active-Center Histidine in Chymotrypsin and Subtilisin Complexed to Peptideboronic Acids without Specific ¹⁵N Labeling¹

Abstract

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].

Tautomerism, acid‐base equilibria, and H‐bonding of the six histidines in subtilisin BPN′ by NMR

“…We confirmed the presence of this H‐bond by discovering and interpreting the anomolously large His C ε1 ‐H 1 H chemical shifts present in α‐lytic protease, subtilisin, and their boronic acid‐inhibited complexes. Similar reports for other serine proteases, including a boronic acid complex of subtilisin E soon followed (Bao et al 1998, 1999; Lin et al 1998). The additional H‐bond suggested to us a novel reaction‐driven imidazole ring flip mechanism that could solve a long‐standing dilemma regarding how such enzymes catalyze both the formation and productive breakdown of the tetrahedral intermediates (Ash et al 2000).…”

Unusual1H NMR chemical shifts support (His) Cɛ1—H⋅⋅⋅O⩵C H-bond: Proposal for reaction-driven ring flip mechanism in serine protease catalysis

“…Consonni et al (44) have demonstrated that these assignments were to autolyzed fragments of enzyme. Bao et al (41) have recently reported a chemical shift of 9.20 ppm for the MeOSuc-Ala-Ala-Pro-boroPhe (AAPbF) complex of subtilisin E. To the best of our knowledge, our finding of a C 1 OH proton chemical shift (9.18 ppm) for a resting, active subtilisin has not been elsewhere reported. Here we also report a C 1 OH proton pH-independent chemical shift for the subtilisin BPNЈ͞AAPbF complex of 9.45 ppm, to our knowledge the most downfield yet observed (Table 1).…”

13C and 1H NMR Studies of Ionizations and Hydrogen Bonding in Chymotrypsin-Glyoxal Inhibitor Complexes

“…The hydrogen-bonded proton at ϳ18 ppm in unligated ␣-chymotrypsin has been assigned to the N ␦1 proton of the imidazolium ion of histidine 57 that is hydrogen-bonded to aspartate 102 (8,10,22). At 4°C this proton titrated from 18.0 Ϯ 0.1 at low pH to 14.7 Ϯ 0.1 at high pH with a pK a of 6.7 Ϯ 0.1 at 4°C.…”