| Abstract
| - To address questions regarding the mechanism of serine protease catalysis, we have solvedtwo X-ray crystal structures of α-lytic protease (αLP) that mimic aspects of the transition states: αLP at pH5 (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-flippingduring the acylation step of the reaction. Rather, our data suggests that upon protonation of His57, Ser195undergoes a conformational change that destabilizes the His57-Ser195 hydrogen bond, preventing theback-reaction. In both structures the His57-Asp102 hydrogen bond in the catalytic triad is a normal ionichydrogen bond, and not a low-barrier hydrogen bond (LBHB) as previously hypothesized. We proposethat the enzyme has evolved a network of relatively short hydrogen bonds that collectively stabilize thetransition states. In particular, a short ionic hydrogen bond (SIHB) between His57 Nε2 and the substrate'sleaving group may promote forward progression of the TI1-to-acylenzyme reaction. We provide experimentalevidence that refutes use of either a short donor−acceptor distance or a downfield 1H chemical shift assole indicators of a LBHB.
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