Crystal versus solution structures of enzymes: NMR spectroscopy of a crystalline serine protease.

The hydrogen-bonding status of His57 in the catalytic triad (Asp-His-Ser) of serine protease has important mechanistic implications for this class of enzymes. Recent nitrogen-15 nuclear magnetic resonance (NMR) studies of alpha-lytic protease find His57 and Ser195 to be strongly hydrogen-bonded, a result that conflicts with the corresponding crystallographic studies, thereby suggesting that the crystal and solution structures may differ. This discrepancy is addressed and resolved in a nitrogen-15 NMR study of the enzyme in the crystalline state. The results show that the His-Ser and Asp-His interactions are identical in crystals and solutions, but that in crystals His57 titrates with a pKa of 7.9, nearly one pKa unit higher than in solution. This elevated pKa accounts for the absence of the His-Ser hydrogen bond in previous x-ray studies.