A Spectroscopic Investigation of S-Trifluoroethylthiopapain. An Investigation of the Active Site of Papain

Abstract

The pH dependence of the ¹⁹F chemical shift and the fluorescence spectrum of S‐2,2,2‐trifluoro1,1‐dideuteroethyl‐thio‐papain are analysed in terms of dependence on the ionisation of aspartic‐acid158 and histidine‐159. The ¹⁹F probe causes negative cooperativity between these groups, and does not detected any ionisation at high pH. The intermediate chemical exchange rates for the ionisation of aspartic‐acid‐158 and histidine‐159 allow the approximate rate constants for proton transfer to be calculated. The rather low rate constants are explained in terms of the hydrophobicity of the activesite region and the net positive charge on the enzyme resulting from its high isoelectric point.