A proton-magnetic-resonance study of N-trifluoroacetyl-L-alanyl-L-phenylalaninal binding to alpha-chymotrypsin

Abstract

Cross-saturation NMR studies have shown that the dipeptide aldehyde N-trifluoroacetyl-L-alanyl-L-phenylalaninal forms a hemiacetal complex with alpha-chymotrypsin. The free aldehyde resonance was seen to broaden upon addition of alpha-chymotrypsin and a detailed analysis has identified the enzyme-bound hemiacetal as the species which was in slow exchange with the free aldehyde and so gave rise to the perturbation. The line broadening was found to be dependent on p2H and this behaviour could be adequately described by the ionisation of a single group on the free enzyme (pKa2H7.6) with the alkaline form being required for hemiacetal formation. The pH dependence of alpha-chymotrypsin-induced catalysis around neutral pH correlates well with the observed p2H dependence of hemiacetal formation, indicating that it is an intermediate in the catalytic mechanism.