title: Mixed disulfide formation at Cys141 leads to apparent unidirectional attenuation of Aspergillus niger NADP-glutamate dehydrogenase activity.
abstract: NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol) resulted in preferential attenuation of AnGDH reductive amination (forward) activity but with a negligible effect on oxidative deamination (reverse) activity, when monitored in the described standard assay. Such a disulfide modified AnGDH displaying less than 1.0% forward reaction rate could be isolated after 2-HED treatment. This unique forward inhibited GDH form (FIGDH), resembling a hypothetical 'one-way' active enzyme, was characterized. Kinetics of 2-HED mediated inhibition and protein thiol titrations suggested that a single thiol group is modified in FIGDH. Two site-directed cysteine mutants, C141S and C415S, were constructed to identify the relevant thiol in FIGDH. The forward activity of C141S alone was insensitive to 2-HED, implicating Cys141 in FIGDH formation. It was observed that FIGDH displayed maximal reaction rate only after a pre-incubation with 2-oxoglutarate and NADPH. In addition, compared to the native enzyme, FIGDH showed a four fold increase in K0.5 for 2-oxoglutarate and a two fold increase in the Michaelis constants for ammonium and NADPH. With no change in the GDH reaction equilibrium constant, the FIGDH catalyzed rate of approach to equilibrium from reductive amination side was sluggish. Altered kinetic properties of FIGDH at least partly account for the observed apparent loss of forward activity when monitored under defined assay conditions. In sum, although Cys141 is catalytically not essential, its covalent modification provides a striking example of converting the biosynthetic AnGDH into a catabolic enzyme.
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India.
authors.forename: Adhish S
authors.initials: AS
authors.lastname: Walvekar
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India.
authors.forename: Rajarshi
authors.initials: R
authors.lastname: Choudhury
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India.
authors.forename: Narayan S
authors.initials: NS
authors.lastname: Punekar
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000737:chemistry / Q000201:enzymology*; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D003641:Deamination; D004220:Disulfides / Q000378:metabolism*; D000431:Ethanol / Q000031:analogs & derivatives* / Q000378:metabolism; D018086:Glutamate Dehydrogenase (NADP+) / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D016415:Sequence Alignment
chemical_list: D004220:Disulfides; D000431:Ethanol; C031319:2-hydroxyethyl disulfide; D018086:Glutamate Dehydrogenase (NADP+); D003545:Cysteine