Thermal denaturation of beta-galactosidase and of two site-specific mutants.
The thermal denaturation of wild-type beta-galactosidase and two beta-galactosidases with substitutions at the active site was studied by kinetics, differential scanning calorimetry, electrophoresis, molecular exclusion chromatography, and circular dichroism. From the results, a model is developed for thermal denaturation of beta-galactosidase which includes the reversible dissociation of ligands, reversible formation of an inactive tetramer, irreversible dissociation of the inactive tetramer to inactive monomers, and subsequent aggregation of inactive monomers to dimers and larger aggregates. Under some conditions, partial reversibility of the activity loss could be demonstrated, and several intermediates in the thermal denaturation process were trapped by quenching and observed by electrophoresis and molecular exclusion chromatography. The ligands Mg2+ and phenylethyl thio-beta-D-galactoside increase the stability of beta-galactosidase to heat denaturation by shifting the ligand binding equilibrium according to Le Chatelier's principle, thus decreasing the concentration of the ligand-free tetramer which can proceed to subsequent steps. Circular dichroism results indicated that beta-galactosidase is dominated by beta-sheet with lower amounts of alpha-helix. Large changes in secondary structure begin to occur only after activity has been lost. Single amino acid changes at the active site can have significant effects on thermal stability of beta-galactosidases. Some of the effects result from increased thermal stability of the ligand-free enzyme itself. Other effects result from changes in ligand binding, but the magnitude of the resulting changes in stability is not related to the strength of ligand binding in a simple fashion.
Edwards
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Division of Biochemistry, Faculty of Science, University of Calgary, Alberta, Canada.
Jacobson
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Huber
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D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D006358:Hot Temperature; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013862:Thiogalactosides / Q000378:metabolism; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D008024:Ligands; D013862:Thiogalactosides; C066907:phenylethylthio-beta-galactopyranoside; D001616:beta-Galactosidase; D008274:Magnesium