Co‐operative Ionisation of Aspartic‐Acid‐158 and Histidine‐159 in Papain

Abstract

The chemical shift of the single resonance in the ¹⁹F nuclear magnetic resonance spectrum of papain which has been irreversibly inhibited by 3‐bromo‐1,1,1‐trifluoropropanone, exhibits pH‐dependence. The fluorescence intensity of this papain derivative shows pH‐dependence on two groups which exhibit co‐operative ionisation. This co‐operative behaviour is probably a function of the probe since the fluorescence intensity of S‐ethane‐thio‐papain is dependent on a single ionisation constant, whereas that of S‐(2‐hydroxyethane)‐thio‐papain is dependent on two ionisable groups again acting co‐operatively. The 1,1,1‐trifluoroketone probe will be hydrated in aqueous solution and would be capable of hydrogen bonding with the protein. The two groups detected are considered to be aspartic‐acid‐158 and histidine‐159. The co‐operative ionisation of these groups in substrate hydrolysis is discussed.