COMPARISON OF INACTIVATION AND CONFORMATIONAL-CHANGES OF AMINOACYLASEDURING DENATURATION IN LITHIUM DODECYL-SULFATE SOLUTIONS

The denaturation of aminoacylase in LDS solutions of different concent rations has been studied by following the changes in the ultraviolet a bsorbance, circular dichroism and intrinsic fluorescence. The results obtained show that the denaturation of the enzyme results in negative peaks at 287 and 295 nm in the denatured minus native enzyme differenc e spectrum. The fluorescence emission intensity of the enzyme decrease s with no red shift of emission maximum in LDS solutions of increasing concentrations. In the LDS concentration regions employed in present study, no marked changes of secondary structure of the enzyme have bee n observed by following the changes in far ultraviolet CD spectra. The inactivation of this enzyme has been followed and compared with the u nfolding observed during denaturation in LDS solutions. A marked inact ivation is already evident at low LDS concentrations before significat ion conformational changes can be detected by ultraviolet absorbance a nd fluorescence changes. The inactivation rate constants of free enzym e and substrate-enzyme complex were determined by the kinetics method of the substrate reaction in the presence of inactivator previously de scribed by Tsou. At the same LDS concentrations, the inactivation rate constants of the enzyme are a order of magnitude faster than the rate constants of conformational changes at least. The above results show that the active sites of metal enzyme containing Zn2+ are also situate d in a limited and flexible region of the enzyme molecule that is more fragile to denaturants than the protein as a whole. (C) 1997 Elsevier Science B.V.