Subangstrom crystallography reveals that short ionic hydrogen bonds, and not a His-Asp low-barrier hydrogen bond, stabilize the transition state in serine protease catalysis

To address questions regarding the mechanism of serine protease catalysis, we have solved two X-ray crystal structures of α-lytic protease (αLP) that mimic aspects of the transition states:  αLP at pH 5 (0.82 A resolution) and αLP bound to the peptidyl boronic acid inhibitor, MeOSuc-Ala-Ala-Pro-boroVal (0.90 A resolution). Based on these structures, there is no evidence of, or requirement for, histidine-flipping during the acylation step of the reaction. Rather, our data suggests that upon protonation of His57, Ser195 undergoes a conformational change that destabilizes the His57-Ser195 hydrogen bond, preventing the back-reaction. In both structures the His57-Asp102 hydrogen bond in the catalytic triad is a normal ionic hydrogen bond, and not a low-barrier hydrogen bond (LBHB) as previously hypothesized. We propose that the enzyme has evolved a network of relatively short hydrogen bonds that collectively stabilize the transition states. In particular, a short ionic hydrogen bond (SIHB) between His57 Ne2 an...