Nitrogen-15 NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism

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Abstract
Nitrogen-15 NMR spectroscopy has been used to study the hydrogen-bonding interactions involving the histidyl residue in the catalytic triad of .alpha.-lytic protease in the resting enzyme and in the transition-state or tetrahedral intermediate analogue complexes formed with phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The 15N shifts indicate that a strong hydrogen bond links the active site histidine and serine residues in the resting enzyme in solution. This result is at odds with interpretations of the X-ray diffraction data of .alpha.-lytic protease and of other serine proteases, which indicate that the serine and histidine residues are too far apart and not properly aligned for the formatin of a hydrogen bond. In addition, the nitrogen-15 shifts demonstrate that protonation of the histidine imidazole ring at low pH in the transition-state or tetrahedral intermediate analogue complexes formed with phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate triggers the disruption of the aspartate-histidine hydrogen bond. These results suggest a catalytic mechanism involving directed movement of the imidazole ring of the active site histidyl residue.