Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Kinetics of reassociation and reactivation from inactive subunits.
Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is denatured in 8 M urea and dissociated into its two inactive subunits. Denaturation leads to an approximately 80% decrease in protein fluorescence and a 20-nm red shift in the emission maximum. Upon dilution, the urea-treated enzyme regains catalytic activity (approximately 70%). The reactivated enzyme is indistinguishable from the native enzyme based on a number of physicochemical and enzymological criteria. The kinetics of renaturation and reactivation were monitored by measuring the rates of regain of native fluorescence and appearance of activity and the accessibility of histidine residues toward diethyl pyrocarbonate modification. Regain of native fluorescence was too rapid to measure at 25 degrees C; at 5 degrees C the initial phase was also too rapid, but a slower phase was monitored and shown to obey first-order kinetics with k = (5.9 +/- 1.3) x 10(-3) s-1. Reappearance of activity was measured at several protein concentrations; reactivation followed second-order kinetics with k = (4.85 +/- 0.47) x 10(-3) M-1 min-1. Reactivation was stimulated to different degrees by either the initial or delayed addition of NAD+, NADP+, or glucose 6-phosphate. During the initial, rapid phase of renaturation, approximately 3 of the enzyme's 12 histidine residues become unreactive toward diethyl pyrocarbonate; concomitant with the subsequent reactivation, approximately 7 more histidines become inaccessible to diethyl pyrocarbonate. The data are consistent with a model for enzyme renaturation and reactivation in which the unfolded subunits rapidly refold to an inactive structure that can dimerize slowly to generate native enzyme. Specific ligands stimulate reactivation by binding to refolded subunits or incompletely folded dimers.
D019298:Glucose-6-Phosphate; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D005958:Glucosephosphates / Q000378:metabolism; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014508:Urea
D005958:Glucosephosphates; D046911:Macromolecular Substances; D009243:NAD; D009249:NADP; D019298:Glucose-6-Phosphate; D014508:Urea; D005954:Glucosephosphate Dehydrogenase