title: Mechanisms of irreversible thermal inactivation of Bacillus alpha-amylases.
abstract: Molecular mechanisms of irreversible thermal inactivation of two bacterial alpha-amylases, from the mesophile Bacillus amyloliquefaciens and from the thermophile Bacillus stearothermophilus, have been elucidated in the pH range of relevance to enzymatic catalysis. At pH 5.0, 6.5, and 8.0, B. amyloliquefaciens alpha-amylase irreversibly inactivates due to a monomolecular conformational process, formation of incorrect (scrambled) structures which subsequently undergo aggregation. At the last pH, this process can be suppressed by the presence of the substrate starch and consequently a covalent process, deamidation of asparagine and/or glutamine residues, becomes the cause of loss of enzymatic activity at 90 degrees C. Monomolecular conformational scrambling is the predominant cause of irreversible inactivation of B. stearothermophilus alpha-amylase at 90 degrees C at pH 5.0, 6.5, and 8.0. At pH 6.5 another contributing inactivation mechanism is the deamidation of amide residues, and at pH 8.0, O2 oxidation of the enzyme's cysteine residue.
authors.affiliation: Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge 02139.
authors.forename: S J
authors.initials: SJ
authors.lastname: Tomazic
authors.forename: A M
authors.initials: AM
authors.lastname: Klibanov
mesh_terms: D000081:Acetamides / Q000494:pharmacology; D000577:Amides; D001216:Asparagine; D001407:Bacillus / Q000201:enzymology*; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004789:Enzyme Activation; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005973:Glutamine; D006207:Half-Life; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013438:Sulfhydryl Compounds; D013816:Thermodynamics; D000516:alpha-Amylases / Q000378:metabolism*
chemical_list: D000081:Acetamides; D000577:Amides; D013438:Sulfhydryl Compounds; D005973:Glutamine; D001216:Asparagine; C030686:acetamide; D000516:alpha-Amylases; D003545:Cysteine