Mechanism of serine protease action. Ionization behavior of tetrahedral adduct between α-lytic protease and tripeptide aldehyde studied by carbon-13 magnetic resonance

Magnetic resonance techniques have been used to study the ionization behavior of the catalytic triad of the serine protease, α-lytic protease, in the tetrahedral, hemiacetal complex it forms with the aldehyde inhibitor, N-ac-L-ala-L-pro-L-alaninal. Chemical shift, coupling constant and relaxation measurements of a carbon-13 nucleus specifically incorporated in C-2 of the imidazole ring of the single histidine residue of the protein show that, above pH 7, the imidazole ring of the catalytic triad in the enzyme + aldehyde complex is neutral. We suggest, further, that a neutral carboxylic acid group for Asp 102 and an oxyanion for the hemiacetal are most likely to describe the state of ionization of the other groups above pH 7. Around pH 6·25, both the oxyanion and the histidine become protonated in a co-operative process which forces the histidine away from its rigidly localized position as a member of the catalytic triad into a solution-like environment