Proton and fluorine nuclear magnetic resonance spectroscopic observation of hemiacetal formation between N-acyl-p-fluorophenylalaninals and .alpha.-chymotrypsin

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Abstract
1H NMR spectroscopy shows that the free aldehyde and not the hydrate of N-acetyl-DL-p-fluorophenylalaninal binds to .alpha.-chymotrypsin. A 1H NMR cross-saturation experiment shows that the initial noncovalent complex is in equilibrium with a hemiacetal formed between the aldehyde and the active site serine residue. 19F NMR spectra of N-acetyl-DL- (and N-acetyl-L-) p-fluorophenylalaninal in the presence of .alpha.-chymotrypsin show separate signals for the hemiacetal complex, the bound aldehyde, the free aldehyde, and the free hydrate. N-Benzoyl-DL-p-fluorophenylalaninal 19F NMR signals are also observed for all species except the bound aldehyde form in the presence of .alpha.-chymotrypsin. The D and L enantiomers of the hydrate of the N-acetyl aldehyde inhibitor give separate 19F NMR signals, arising from chemical exchange between the L-aldehyde-.alpha.-chymotrypsin complex and the free L hydrate. The enzyme-bound inhibitor 19F signals disappear upon proton decoupling due to a negative nuclear Overhauser effect. Upon gated decoupling of protons, the L hydrate and free aldehyde 19F signals are reduced in intensity relative to that of the D hydrate signal in the racemate aldehyde complex. This is attributed to a saturation transfer of the heteronuclear nuclear Overhauser effect.

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