Reversible pH-Induced Dissociation of Glucose Dehydrogenase from Bacillus megaterium II. Kinetics and Mechanism

Abstract

Glucose dehydrogenase from Bacillus megaterium exists as a stable, active tetram er at pH 6.5. By shifting the pH to 9, the enzyme is, completely and reversibly, dissociated into four inactive protom ers. Kinetics and mechanism of this pH-induced dissociation have been studied, at various enzyme concentrations, by ultraviolet absorption, circular dichroism, normal and stopped-flow fluorescence as well as by light scattering andactivity measurements. Dissociation of the fully active tetram er proceeds via three distinct kinetic steps: (1) fast conformational rearrangem ent of the tetram er, without any loss of activity (t12 0.0075 sec);(2) slow isomerization to a tetram er with lower specific activity (t12 27 sec); (3) subsequent dissociation of this rearranged tetram er into inactive m onom ers (t12 114 sec) with still intact native secondary structure. All three processes follow first-order kinetics. Both rate and extent of the dissociation are reduced, with a concomitant shift to higher reaction orders, by increasing the NaCl concentration in the buffer. This suggests the establishment of a dissociation/association equilibrium, due to the concentrationdependent stabilization ofthe tetram eric enzyme state by NaCl. © 1987, Walter de Gruyter. All rights reserved.