title: Oxidation-reduction properties of two engineered redox-sensitive mutant Escherichia coli malate dehydrogenases.
abstract: Redox potentials for two inactivating intrasubunit disulfides that link helix-5 and helix-9 in mutant Escherichia coli malate dehydrogenases have been determined. The Em is -285 mV when cysteines are at positions 121 and 305 and -295 mV when the cysteines are at positions 122 and 305. Oxidation to the disulfide affects kcat but not Km values. In the single V121C and N122C mutants, the Cys in helix-5 affects the Km for oxalacetate. The pH optimum in the direction of malate formation is affected by the redox state of the enzyme. Clearly, a disulfide bond can and does form between Cys residues substituted into positions 121 or 122 in the nucleotide binding domain and 305 in the carbon substrate binding domain of this NAD-dependent malate dehydrogenase. Apparently, crosslinking the domains interferes with catalysis.
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409, USA.
authors.forename: A
authors.initials: A
authors.lastname: Setterdahl
authors.forename: M
authors.initials: M
authors.lastname: Hirasawa
authors.forename: L M
authors.initials: LM
authors.lastname: Bucher
authors.forename: C A
authors.initials: CA
authors.lastname: Dholakia
authors.forename: P
authors.initials: P
authors.lastname: Jacquot
authors.forename: H
authors.initials: H
authors.lastname: Yards
authors.forename: F
authors.initials: F
authors.lastname: Miller
authors.forename: F J
authors.initials: FJ
authors.lastname: Stevens
authors.forename: D B
authors.initials: DB
authors.lastname: Knaff
authors.forename: L E
authors.initials: LE
authors.lastname: Anderson
mesh_terms: D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D009243:NAD / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004220:Disulfides; D010940:Plant Proteins; D011994:Recombinant Proteins; D009243:NAD; D008291:Malate Dehydrogenase; D003545:Cysteine; D010100:Oxygen