A kinetic and fluorimetric investigation of papain modified at tryptophan-69 and -177 by N-bromosuccinimide.
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 pK(a) of the acidic limb of the pH profile by more than 1 pH unit for both k(cat.) and k(cat.)/K(m). 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 pK(a) 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 pK(a) 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 S(2) and S(1)' subsites respectively.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008968:Molecular Conformation; D010084:Oxidation-Reduction; D010206:Papain / Q000032:analysis; D013050:Spectrometry, Fluorescence; D013388:Succinimides; D013438:Sulfhydryl Compounds / Q000032:analysis; D014364:Tryptophan / Q000032:analysis
D000596:Amino Acids; D013388:Succinimides; D013438:Sulfhydryl Compounds; D006639:Histidine; D014364:Tryptophan; D010206:Papain