| The denaturation of the dimeric enzyme glucose-6-phosphate dehydrogenase (G6PD) from Leuconostoc mesenteroides by guanidine hydrochloride (GdnHCl) was studied in detail for the first time using enzymatic activity, light scattering, intrinsic fluorescence, and circular dichroism measurements. Equilibrium experiments revealed that between 0.9 and 1.2 M GdnHCl 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. The inactive, partially unfolded, dimeric intermediate which formed was highly susceptible to aggregation, especially upon removal of the GdnHCl by dilution, probably due to exposure of "sticky" hydrophobic stretches of the polypeptide chain. Kinetics experiments demonstrated that several transient denaturation intermediates also form.;In 4 M GdnHCl, G6PD dissociated to subunits and was extensively unfolded. Removal of this high GdnHCl concentration by dilution allowed G6PD to renature as measured by enzyme reactivation, but the reactivation yield only reached 55%. The remaining 45% of the enzyme aggregated and precipitated out of solution, which could not be prevented by stabilizing additives. Based on the enzyme concentration dependence of the reactivation yield and measurements of the aggregation rate versus the reactivation rate, it was determined that aggregation competes kinetically with reactivation for a partially folded intermediate only very early in the process, during the rapid GdnHCl dilution step.;The kinetics of reactivation were sigmoidal, indicating that more than one rate-limiting reaction was involved, and were dependent on enzyme concentration in a higher than first order manner, indicating that association of subunits is one of the rate-limiting reactions. A compatible renaturation mechanism involves a bi-unimolecular (subunit association-folding) reaction sequence, with rate constants equal to 2.10... |
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