Unusual H-1 NMR chemical shifts support (His) C-epsilon 1-H center dot center dot center dot O = C H-bond: Proposal for reaction-driven ring flip mechanism in serine protease catalysis

C-13-selective NMR. combined with inhibitor perturbation experiments, shows that the C-epsilon 1-H proton of the catalytic histidine in resting ol-lyt ic protease and subtilisin BPN' resonates, when protonated. at 9.22 ppm and 9.18 ppm, respectively, which is outside the normal range for such protons and approximate to 0.6 to 0.8 ppm further downfield than previously report ed. They also show that the previous or-lytic protease assignments [Markley , J. L., Neves, D. E., Westler. W. M., Ibanez, I. B., Porubcan, M. A. & Bai llargeon, M. W. (1980) Front Protein Chem. 10, 31-61] were to signals from inactive or denatured protein. Simulations of linewidth vs. pH demonstrate that the true signal is more difficult to detect than corresponding signals from inactive derivatives, owing to higher imidazole pK(a) values and larg er chemical shift differences between protonated and neutral forms. A compi lation and analysis of available NMR data indicates that the true C-epsilon 1-H signals from other serine proteases are similarly displaced downfield, with past assignments to more upfield signals probably in error. The downf ield displacement of these proton resonances is shown to be consistent with an H-bond involving the histidine C-epsilon 1-H as donor, confirming the o riginal hypothesis of Derewenda et al. [Derewenda. Z. S., Derewenda, U. & K obos, P. M. (1994) J. Mol. Biol. 241, 83-93], which was based on an analysi s of literature x-ray crystal structures of serine hydrolases. The invariab ility of this H-bond among enzymes containing Asp-His-Ser triads indicates functional importance. Here, we propose that it enables a reaction-driven i midazole ring flip mechanism, overcoming a major dilemma inherent in all pr evious mechanisms, namely how these enzymes catalyze both the formation and productive breakdown of tetrahedral intermediates.