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

Abstract

Proton nuclear magnetic resonance (NMR) spectroscopy shows that the free aldehyde and not the hydrate of N-acetyl-DL-p-fluorophenylalaninal binds to alpha-chymotrypsin. A proton 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. Fluorine 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 fluorine 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 fluorine NMR signals, arising from chemical exchange between the L-aldehyde-alpha-chymotrypsin complex and the free L hydrate. The enzyme-bound inhibitor fluorine signals disappear upon proton decoupling due to a negative nuclear Overhauser effect. Upon gated decoupling of protons, the L hydrate and free aldehyde fluorine 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.