Solid-State O NMR of Unstable Acyl-Enzyme Intermediates: A Direct Probe of Hydrogen Bonding Interactions in the Oxyanion Hole of Serine Proteases.

We report preparation, trapping, and solid-state O NMR characterization of three unstable acyl-enzyme intermediates (≈ 26 kDa): p-N,N-dimethylamino-[O]benzoyl-chymotrypsin, trans-o-methoxy-[O]cinnamoyl-chymotrypsin, and trans-p-methoxy-[O]cinnamoyl-chymotrypsin. We show that both the O chemical shifts and nuclear quadrupolar parameters obtained for these acyl-enzyme intermediates in the solid state are correlated with their deacylation rate constants measured in aqueous solution. With the aid of quantum mechanical calculations, the experimental O NMR parameters were interpreted as to reflect the hydrogen bonding interactions between the carbonyl (C═O) functional group of the acyl moiety and the two NH groups from the protein backbone (Ser195 and Gly193) in the oxyanion hole, a general feature of all serine proteases. Our results further suggest that the O chemical shift and quadrupole coupling constant display distinctly different sensitivities toward different aspects of hydrogen bonding, such as hydrogen bond distance and direction. This work demonstrates the utility of O as a useful nuclear probe in NMR studies of enzymes.