Renaturation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides after denaturation in 4 M guanidine hydrochloride: kinetics of aggregation and reactivation.
In 4 M guanidine hydrochloride (GdnHCl), the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (G6PD) dissociated to subunits and was extensively unfolded. Rapid dilution of this high GdnHCl concentration allowed G6PD to partially renature, as measured by enzyme reactivation, to a level which depended on the conditions employed. The fraction of the enzyme which did not renature aggregated and precipitated out of solution, a process which could not be substantially prevented by stabilizing additives. Based on the enzyme concentration dependence of the reactivation yield and on a comparison of the aggregation and reactivation rates, it was determined that aggregation and reactivation compete kinetically for a partially-folded intermediate only very early in the process, during the rapid GdnHCl-dilution step. The kinetics of G6PD reactivation were sigmoidal, indicating that this process involves more than one rate-limiting reaction. The kinetics depended on enzyme concentration in a higher than first-order manner, indicating that association of subunits is one of the rate-limiting reactions. A renaturation mechanism compatible with these observations is described, which involves a bi-unimolecular (subunit association-folding) reaction sequence, with rate constants equal to 2.19 microM-1 min-1 and 0.140 min-1, respectively. This mechanism involves an inactive, dimeric, G6PD-folding intermediate, a species whose existence has recently been established by equilibrium denaturation experiments (Plomer, J.J. and Gafni, A. (1992) Biochim. Biophys. Acta 1122, 234-242).
Plomer
J J
JJ
Institute of Gerontology, University of Michigan, Ann Arbor 48109.
Gafni
A
A
D002021:Buffers; D004793:Enzyme Reactivators; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D008956:Models, Chemical; D011487:Protein Conformation; D011489:Protein Denaturation; D013997:Time Factors
D002021:Buffers; D004793:Enzyme Reactivators; D006146:Guanidines; D005954:Glucosephosphate Dehydrogenase; D019791:Guanidine