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.
Sharkey
Michael A
MA
UCD School of Biomolecular and Biomedical Sciences, Conway Institute, University College Dublin, Belfield, Dublin, Republic of Ireland.
Engel
Paul C
PC
D003001:Cloning, Molecular; D003067:Coenzymes / Q000032:analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D003067:Coenzymes; D011994:Recombinant Proteins; D005969:Glutamate Dehydrogenase