The p K a value of the active site histidine in photo‐oxidised papain

It has recently been suggested that the active site histidine residue in papain and stem-bromelain have pK, values of 6.7 and 6.4 respectively and since these enzymes are essentially pH-independent for substrate hydrolysis at these pH values, histidine is not directly involved in their catalytic mechanism [l-3] This conclusion is based on photo-oxidation experiments during which the active site cysteine residue was oxidised, but this could be reversed by dithiothreitol and the thiol titre of the enzyme completely restored. What was not clear from these experiments, however, was the relative rates of photo-oxidation of the various residues. If, as appeared likely, the thiol group was photo-oxidised more rapidly than the imidazole group, the pK, derived from the pH-dependence curve for the loss of histidine would clearly not be that of the native enzyme. In order to test this possibility, papain was photo-oxidised under conditions described by Okumara and Murachi [3] and the pH-dependence of the fluorescence emission intensity of the product determined. Tryptophan-177 in papain is responsible for almost half the fluorescence intensity of the enzyme at high pH, and when the thiol group is blocked, the pH-dependence of its fluorescence intensity reflects the state of ionisation of the active site histidine residue [4].