Stability of Arthrobacter D-xylose isomerase to denaturants and heat.
There was no inactivation of Mg(2+)-containing Arthrobacter D-xylose isomerase up to 1 h in 0-8 M-urea at 22 degrees C, but over this range there was rapid reversible dissociation into fully active dimers with a midpoint around 4 M-urea, as shown by gradient urea gels with an activity stain, and by ion-exchange chromatography and gel filtration in urea buffers. These dimers must have the A-B* conformation, since the tetramer could dissociate into A-A*, A-B or A-B* dimer conformations, but only residues across the A-B* interface contribute to the active site. The kinetics of inactivation of the Mg(2+)-containing enzyme in 8 M-urea at higher temperatures suggest a partially unfolded Mg-A-B* dimer intermediate with 50% activity, followed by irreversible inactivation coincident with the appearance of unfolded monomer. In 0-4 M guanidinium chloride, a similar reversible dissociation into active dimers occurs, but activity falls, suggesting that A-A* and/or A-B dimers might be part of the mixture. Low concentrations of SDS also give active dimers leading to unfolded monomers, but SDS above 1% (w/v) provides relative stabilization. The apoenzyme is least thermostable (t 1/2 at 80 degrees C, pH 7, = 0.06 h) but Mg2+ stabilizes strongly (t 1/2 = 5.5 h) and Co2+ even more so. Competitive inhibitors or substrates provide a small further stabilization, but this effect is more marked at 80 degrees C, pH 5.5. Together with a marked decrease in optimum pH with temperature, this allows batch isomerizations of glucose under these conditions that produce clean but sweeter syrups.
Rangarajan
M
M
Centre for Biotechnology, Imperial College of Science, Technology and Medicine, London, U.K.
Asboth
B
B
Hartley
B S
BS
D019747:Aldose-Ketose Isomerases; D001173:Arthrobacter / Q000201:enzymology*; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005632:Fructose / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000494:pharmacology; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D046911:Macromolecular Substances; D015636:Magnesium Chloride; D005632:Fructose; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D019791:Guanidine