Apparent negative co-operativity and substrate inhibition in overexpressed glutamate dehydrogenase from Escherichia coli

The gene for Escherichia coli glutamate dehydrogenase (EcGDH) has been overexpressed, and a simplified purification procedure afforded greatly increased yields of c. 40 mg pure EcGDH L⁻¹ culture. EcGDH was unstable at a low concentration in plastic tubes, but stabilization measures allowed a robust kinetic characterization. Contrary to past reports, EcGDH deviates from Michaelis–Menten kinetics, exhibiting apparent mild negative co-operativity with both l -glutamate and NADP⁺, with Hill coefficients of 0.90 and 0.92, respectively. NADPH yielded simple Michaelis–Menten kinetics but both 2-oxoglutarate and NH₄⁺ showed substrate inhibition. pH optima were 9 for oxidative deamination and 8 for reductive amination.