Observation of a Short, Strong Hydrogen Bond in the Active Site of Hydroxynitrile Lyase from Hevea brasiliensis Explains a Large pKa Shift of the Catalytic Base Induced by the Reaction Intermediate

The hydroxynitrile lyase from Hevea brasiliensis ( Hb HNL) uses a catalytic triad consisting of Ser 80 -His 235 -Asp 207 to enhance the basicity of Ser 80 -Oγ for abstracting a proton from the OH group of the substrate cyanohydrin. Following the observation of a relatively short distance between a carboxyl oxygen of Asp 207 and the Nδ 1 (His 235 ) in a 1.1 à crystal structure of Hb HNL, we here show by 1 H and 15 N-NMR spectroscopy that a short, strong hydrogen bond (SSHB) is formed between the two residues upon binding of the competitive inhibitor thiocyanate to Hb HNL: the proton resonance of H-Nδ1(His 235 ) moves from 15.41 ppm in the free enzyme to 19.35 ppm in the complex, the largest downfield shift observed so far upon inhibitor binding. Simultaneously, the D/H fractionation factor decreases from 0.98 to 0.35. In the observable pH range, i.e. between pH 4 and 10, no significant changes in chemical shifts (and therefore hydrogen bond strength) were observed for free Hb HNL. For the complex with thiocyanate, the 19.35 ppm signal returned to 15.41 ppm at ∼pH 8, which indicates a p K a near this value for the H-Nϵ 2 (His 235 ). These NMR results were analyzed on the basis of finite difference Poisson-Boltzmann calculations, which yielded the relative free energies of four protonation states of the His 235 -Asp 207 pair in solution as well as in the protein environment with and without bound inhibitor. The calculations explain all the NMR features, i.e. they suggest why a short, strong hydrogen bond is formed upon inhibitor binding and why this short, strong hydrogen bond reverts back to a normal one at ∼pH 8. Importantly, the computations also yield a shift of the free energy of the anionic state relative to the zwitterionic reference state by about 10.6 kcal/mol, equivalent to a shift in the apparent p K a of His 235 from 2.5 to 10. This huge inhibitor-induced increase in basicity is a prerequisite for His 235 to act as general base in the Hb HNL-catalyzed cyanohydrin reaction.