INACTIVATION BEFORE SIGNIFICANT CONFORMATIONAL CHANGE DURING DENATURATION OF PAPAIN BY GUANIDINE-HYDROCHLORIDE

During denaturation by GuHCl, papain shows a rapid decrease in activit y with increasing concentrations of the denaturant followed by an inte rmediate stage of relatively little change from 1 to 2 M before comple te inactivation at 4 M GuHCl. At GuHCl concentrations lower than 2 M, enzyme activity is more sensitive to GuHCl than noticeable conformatio n changes as followed by fluorescence and CD measurements. Kinetics of GuHCl inactivation were studied by following the substrate reaction i n the presence of denaturant and the apparent rate constants thus obta ined were found to be only slightly higher than those for conformation al changes. However, apparent inactivation rate constants obtained in the presence of saturating concentration of substrate are actually ina ctivation constants for the ES complex. The inactivation rates at diff erent substrate concentrations were, therefore, followed and the micro scopic inactivation rate constants for the free enzyme obtained (Tsou, C.L. (1988) Adv. Enzymol. 61, 381-436). It was found that substrate p rotects strongly against inactivation and at the same GuHCl concentrat ion, the inactivation rate of the free enzyme is 100-fold higher than that of unfolding. The above results show that the activity of papain is more sensitive to GuHCl than its overall conformation and like the enzymes previously studied in this laboratory, its active site is more flexible than the enzyme molecule as a whole.