title: Disulfide bonds are necessary for structure and activity in Aspergillus ficuum phytase.
abstract: The function of disulfide bonds in Aspergillus ficuum phytase was elucidated by unfolding studies, using guanidinium hydrochloride (Gu.HCl) as denaturant. Although the enzyme is totally inactivated by 0.8 M Gu.HCl, at pH 5.0, the active conformation is instantaneously restored by 0.6 M Gu.HCl, at pH 5.0. Conditions which would permit refolding of phytase are completely negated by 10 mM beta-mercaptoethanol and causes its catalytic demise at pH 7.5. Assay of free thiols using Ellman's reagent indicates that none of the thiols in the ten cysteines in phytase are free; five disulfide bonds were predicted for the enzyme. Sequence comparison of mold phytases and yeast acid phosphatases indicates four conserved cysteines. Thus, disulfide bonds play an important role in the folding of fungal phytase; any perturbation of the process of its formation causes an altered three-dimensional structure that is inconsistent with catalytic activity.
authors.affiliation: Southern Regional Research Center, ARS, USDA, New Orleans, Louisiana 70124, USA.
authors.forename: A H
authors.initials: AH
authors.lastname: Ullah
authors.forename: E J
authors.initials: EJ
authors.lastname: Mullaney
mesh_terms: D010832:6-Phytase / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000032:analysis
chemical_list: D004220:Disulfides; D006146:Guanidines; D013438:Sulfhydryl Compounds; D008623:Mercaptoethanol; D004228:Dithionitrobenzoic Acid; D010832:6-Phytase; D019791:Guanidine