On the inactivity of thiol-subtilisin: The role of the intramolecular electric field

Based on computed proton affinities for several model systems, the energetics of proton transfer and the acidity of the catalytic triads Cys-His-Asn (papain). Cys-His-Asp (thiol-subtilisin) and Ser-His-Asp (subtilisin) are discussed. It is shown that in papain the ion-pair Cys−-HisH+ exists owing to the intramolecular electric field, and that a similar situation is found in thiol-subtilisin. but not in subtilisin. Assuming similar reaction mechanisms for papain and thiol-subtilisin - i.e. proton transfer from HisH+ to the NH group of the scissile peptide bond - the inactivity of thil-subtilisin towards proteins is explained by the much greater basicity of His in the complex His-Asp− than in His-Asn. In order for this explanation to be consistent, it is tentatively concluded that the catalytic mechanism of the serine proteases is different from that of the cystein proteases, and involves direct transfer of the serine proton to the leaving group in the acylation step.