A kinetic and fluorimetric investigation of papain modified at tryptophan -69 and -177 by N–bromosuccinimide

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Abstract
A systematic study of the modification of papain (its thiol group protected as a disulphide with mercaptoethanol) by N-bromosuccinimide, showed that 2 molar equiv. modified tryptophan-69 and 4 molar equiv. modified tryptophan -69 and -177. The Michaelis parameters for the catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester by these modified enzymes were determined. The enzymic activity of the modified enzymes was not seriously impaired, but modification of tryptophan-177 raised the apparent pKa of the acidic limb of the pH profile by more than 1 pH unit for both kcat. and kcat./Km. The fluorescence spectra (excitation at 288nm) of the modified enzymes showed that tryptophan -69 contributed about 8% to the fluorescence intensity, whereas tryptophan-177 contributed about 46% at neutral pH. However, the contribution of tryptophan -177 was quenched at low pH and its fluorescence intensity showed sigmoidal pH-dependence, with an apparent pKa of 4.2. Histidine -159, which is in close contact with tryptophan -177, is considered to be the residue responsible for the fluorescence quenching. When tryptophan -177 was modified, presumably generating a less hydrophobic micro-environment, the apparent pKa determined kinetically was raised to about 5.4. By comparing the Michaelis parameters of native papain, papain modified at tryptophan-69 and papain modified at tryptophan-69 and -177 with N–benzyloxycarbonylglycylglycine amide and N–benzyloxycarbonylglycyltryptophan amide, tryptophan-69 and tryptophan -177 were shown to be structural features of the S2 and S1′ subsites respectively.

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