DENATURATION OF GLUCOSE-6-PHOSPHATE-DEHYDROGENASE FROM LEUCONOSTOC-MESENTEROIDES BY GUANIDINE-HYDROCHLORIDE - IDENTIFICATION OF INACTIVE, PARTIALLY UNFOLDED, DIMERIC INTERMEDIATES

The denaturation of the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc, mesenteroides by guanidine hydrochloride has been studied using enzymatic activity, intrinsic fluorescence, circular dichroism, and light scattering measurements. Equilibrium experiments at 25-degrees-C revealed that between 0.9 and 1.2 M denaturant the enzyme underwent a conformational. change, exposing tryptophan residues to solvent, with some loss of secondary structure and a complete loss of enzymatic activity but without dimer dissociation to subunits. This inactive, partially unfolded, dimeric intermediate was susceptible to slow aggregation, perhaps due to exposure of 'sticky' hydrophobic stretches of the polypeptide chain. A second equilibrium transition, reflecting extensive unfolding and dimer dissociation, occurred only at denaturant concentrations above 1.4 M. Kinetics experiments demonstrated that in the denaturant concentration range of 1.7-1.9 M the fluorescence change occurred in two distinct steps. The first step involved a large, very rapid drop in fluorescence whose rate was strongly dependent on the denaturant concentration. This was followed by a small, relatively slow rise in the emission intensity, the rate of which was independent of denaturant concentration. Enzymatic activity was lost with a denaturant-concentration-dependent rate, which was approx. 3-times slower than the rate of the first step in fluorescence change. A denaturation mechanism incorporating several unfolding intermediates and which accounts for all the above results is presented and discussed. While the fully unfolded enzyme regained up to 55% of its original activity upon dilution of denaturant to a concentration that would be expected to support native enzyme, denaturation intermediates were able to reactivate only minimally and in fact were found to aggregate and precipitate out of solution.