title: The Role of Cysteine Residues in Catalysis of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis.
abstract: Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis, can persist in macrophages for decades, maintaining its basic metabolic activities. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is a key player in central carbon metabolism regulation. In replicating MTb, Pck is associated with gluconeogenesis, but in non-replicating MTb, it also catalyzes the reverse anaplerotic reaction. Here, we explored the role of selected cysteine residues in function of MTb Pck under different redox conditions. Using mass spectrometry analysis we confirmed formation of S-S bridge between cysteines C391 and C397 localized in the C-terminal subdomain. Molecular dynamics simulations of C391-C397 bridged model indicated local conformation changes needed for formation of the disulfide. Further, we used circular dichroism and Raman spectroscopy to analyze the influence of C391 and C397 mutations on Pck secondary and tertiary structures, and on enzyme activity and specificity. We demonstrate the regulatory role of C391 and C397 that form the S-S bridge and in the reduced form stabilize Pck tertiary structure and conformation for gluconeogenic and anaplerotic reactions.
authors.affiliation: Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic.
authors.forename: Iva
authors.initials: I
authors.lastname: Machová
authors.affiliation: Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Hubálek
authors.affiliation: Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Lepšík
authors.affiliation: Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic.
authors.forename: Lucie
authors.initials: L
authors.lastname: Bednárová
authors.affiliation: Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic.|Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Prague, Czech Republic.
authors.forename: Markéta
authors.initials: M
authors.lastname: Pazderková
authors.affiliation: Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Prague, Czech Republic.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Kopecký
authors.affiliation: Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Snášel
authors.affiliation: Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Dostál
authors.affiliation: Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic.
authors.forename: Iva
authors.initials: I
authors.lastname: Pichová
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D055162:Biocatalysis; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D004220:Disulfides; D050505:Mutant Proteins; D019764:Phosphoenolpyruvate Carboxykinase (ATP); D003545:Cysteine