Open AccessApparent negative co‐operativity and substrate inhibition in overexpressed glutamate dehydrogenase from Escherichia coli
Author(s) -
Sharkey Michael A.,
Engel Paul C.
Publication year - 2008
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2008.01086.x
Subject(s) - oxidative deamination , glutamate dehydrogenase , substrate (aquarium) , escherichia coli , deamination , kinetics , chemistry , reductive amination , biochemistry , enzyme kinetics , dehydrogenase , amination , enzyme , glutamate receptor , stereochemistry , biology , gene , active site , catalysis , ecology , receptor , physics , quantum mechanics
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 NH 4 + showed substrate inhibition. pH optima were 9 for oxidative deamination and 8 for reductive amination.