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.
Highlights
Glutamate dehydrogenase (GDH) catalyzes reversible reductive amination of 2-oxoglutarate to form L-glutamate using NAD(P) as a cofactor [1]
We report the characterization of this practically one-way active enzyme and have identified the cysteine residue modified in FIGDH, by site-directed mutagenesis
Apparent one-way inactivation of A. niger NADP-GDH Pure preparations of AnGDH when stored at 4uC showed timedependent loss of forward activity while retaining full activity in the reverse direction
Summary
Glutamate dehydrogenase (GDH) catalyzes reversible reductive amination of 2-oxoglutarate to form L-glutamate using NAD(P) as a cofactor [1]. A range of GDH activity responses have been recorded upon thiol modification, that depend on the nature of the reagent and the target site. The reactive cysteines in GDHs from diverse sources have been identified with the use of affinity labels [2,3,4] and thiol specific reagents [5,6,7,8,9]. Cys323 in human GDH isozymes (hGDH1 and hGDH2) is so far the only reactive cysteine identified as important for catalysis [9]. Another cysteine residue, Cys119, is shown to be ADP-ribosylated in both these isozymes. The physiological significance of this modification is being explored currently [10]
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