Unfolding kinetics of glutathione reductase from cyanobacterium Spirulina maxima.
The kinetics of the irreversible unfolding of glutathione reductase (NAD[P]H:GSSG oxidoreductase, EC 1.6.4.2.) from cyanobacterium Spirulina maxima was studied at pH 7.0 and room temperature. Denaturation was induced by guanidinium chloride and the changes in enzyme activity, aggregation state, and tertiary structure were monitored. No full reactivation of enzyme was obtained, even after very short incubation times in the presence of denaturant. Reactivation plots were complex, showing biphasic kinetics. A very fast early event in the denaturation pathway was the dissociation of tetrameric protein into reactivatable native-like dimers, followed by its conversion into a nonreactivatable intermediary, also dimeric. In the final step of the unfolding pathway the latter was dissociated into denatured monomers. Fluorescence measurements revealed that denaturation of S. maxima glutathione reductase is a slow process. Release of the prostethic group FAD was previous to the unfolding of the enzyme. No aggregated species were detected in the unfolding pathway, dismissing the aggregation of denatured polypeptide chains as the origin of irreversibility. Instead, the transition between the two dimeric intermediates is proposed as the cause of irreversibility in the denaturation of S. maxima glutathione reductase. A value of 106.6 +/- 3 kJ mol(-1) was obtained for the activation free energy of unfolding in the absence of denaturant. No evidence for the native monomer in the unfolding pathway was obtained which suggests that the dimeric nature of glutathione reductase is essential for the maintenance of the native subunit conformation.
Rendón
J L
JL
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de Mexico, DF. jrendon@bq.unam.mx
Mendoza-Hernández
G
G
D001426:Bacterial Proteins; D002850:Chromatography, Gel; D000458:Cyanobacteria / Q000201:enzymology*; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005980:Glutathione Reductase / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation / Q000187:drug effects / Q000502:physiology; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D053545:Spirulina; D013696:Temperature; D013816:Thermodynamics
D001426:Bacterial Proteins; D005182:Flavin-Adenine Dinucleotide; D005980:Glutathione Reductase; D019791:Guanidine