Crystal Versus Solution Structures of Enzymes: NMR Spectroscopy of a Crystalline Serine Protease

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Abstract
The hydrogen-bonding status of His 57 in the catalytic triad (Asp-His-Ser) of serine proteases has important mechanistic implications for this class of enzymes. Recent nitrogen-15 nuclear magnetic resonance (NMR) studies of α-lytic protease find His 57 and Ser 195 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 His 57 titrates with a pK a of 7.9, nearly one pK a unit higher than in solution. This elevated pK a accounts for the absence of the His-Ser hydrogen bond in previous x-ray studies.