Comparison of inactivation and unfolding of methanol dehydrogenase during denaturation in guanidine hydrochloride and urea

The activity and the conformational changes of methanol dehydrogenase (MDH) , a quinoprotein containing pyrrolo-quinoline quinone as its prosthetic gro up, have been studied during denaturation in guanidine hydrochloride (GdnHC l) and urea. The unfolding of MDH was followed using the steady-slate and t ime resolved fluorescence methods. Increasing the denaturant concentration in the denatured system significantly enhanced the inactivation and unfoldi ng of MDH. The enzyme was completely inactivated at 1 M GdnHCl or 6 M urea. The fluorescence emission maximum of the native enzyme was at 332 nm. With increasing denaturant concentrations, the fluorescence emission maximum re d-shifted in magnitude to a maximum value (355 nm) at 5 M GdnHCl or 8 M ure a. Comparison of inactivation and conformational changes during denaturatio n showed that in general accord with the suggestion made previously by Tsou , the active sites of MDH are situated in a region more flexible than the m olecule as a whole. (C) 2000 Elsevier Science Ltd. All rights reserved.