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 Å resolution) and αLP bound to the peptidyl boronic acid inhibitor, MeOSuc-Ala-Ala-Pro-boroVal
(0.90 Å 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 Nε2 and the substrate's
leaving group may promote forward progression of the TI1-to-acylenzyme reaction. We provide experimental
evidence that refutes use of either a short donor−acceptor distance or a downfield 1H chemical shift as
sole indicators of a LBHB.