title: The cysteines of catalase HPII of Escherichia coli, including Cys438 which is blocked, do not have a catalytic role.
abstract: Site-directed mutagenesis of the katE gene of Escherichia coli was used to change, individually and in combination, Cys438 and Cys669 to serine in catalase HPII. The Cys438-->Ser mutation caused a 30% reduction in the specific activity of the enzyme, whereas the Cys669-->Ser mutation did not affect enzyme activity. The titration of free sulfhydryl groups in HPII revealed that Cys669 was reactive whereas Cys438 was unreactive. Properties of the modification on Cys438 included alkali lability, insensitivity to methylamine, hydroxylamine or reducing agents, and a mass determined by mass spectrometry to be approximately 43 +/- 2 Da. A hemithioacetal structure is consistent with these properties. Although free sulfhydryl groups do not play a significant role in the stability or catalytic mechanism of HPII, the sulfhydryl agent 2-mercaptoethanol caused a 50% inactivation of HPII along with an irreversible change in the absorption spectrum of the protein. Other sulfhydryl agents, including dithiothreitol, cysteine and glutathione, and the organic peroxide, t-butylhydroperoxide, which cannot directly access the active site, do not affect HPII activity, but they do cause a small reversible change in the absorption spectrum, possibly by a mechanism involving superoxide.
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Canada.
authors.forename: M S
authors.initials: MS
authors.lastname: Sevinc
authors.forename: W
authors.initials: W
authors.lastname: Ens
authors.forename: P C
authors.initials: PC
authors.lastname: Loewen
mesh_terms: D001483:Base Sequence; D002374:Catalase / Q000737:chemistry* / Q000502:physiology; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010545:Peroxides / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D020122:tert-Butylhydroperoxide
chemical_list: D010545:Peroxides; D013439:Sulfhydryl Reagents; D020122:tert-Butylhydroperoxide; D002374:Catalase; D003545:Cysteine