1.1.1.363 | denatured in 8 M urea and dissociated into its two inactive subunits (MW 50000 Da). Denaturation leads to an approximately 80% decrease in protein fluorescence and a 20-nm red shift in the emission maximum. Upon dilution, the urea-treated enzyme regains catalytic activity (approximately 70%). The reactivated enzyme is indistinguishable from the native enzyme based on a number of physicochemical and enzymological criteria. The kinetics of renaturation and reactivation are monitored. Reactivation is stimulated to different degrees by either the initial or delayed addition of NAD+, NADP+, or glucose 6-phosphate. During the initial, rapid phase of renaturation, approximately 3 of the enzyme's 12 histidine residues become unreactive toward diethyl pyrocarbonate; concomitant with the subsequent reactivation, approximately 7 more histidines become inaccessible to diethyl pyrocarbonate |
721570 |