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Properties of phosphorylated NADP+-specific glutamate dehydrogenase fromEscherichia coli

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Abstract

The NADP+-specific glutamate dehydrogenase (GDH) fromEscherichia coli strain D5H3G7, an enzyme that catalyzes the interconversion of α-ketoglutarate andl-glutamate, has been shown to be phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein is extremely acid labile and is unstable at high pH. Treatment of GDH with diethyl pyrocarbonate (DEP), a histidine-modifying reagent, blocked the incorporation of32P from [γ-32P]ATP. GDH catalytic activity was also inhibited by DEP treatment. Hydroxylamine, a reagent hydrolyzing phosphoramidates, catalyzed the removal of phosphate from phosphorylated GDH, suggesting that GDH may be phosphorylated at a histidine residue(s). A total enzymatic hydrolysis of phosphorylated GDH, which was electroeluted from a native polyacrylamide gel, was analyzed by a Dowex 1-8X anion exchange chromatography. The presence of32P-labeled 3-phosphohistidine, characterized and identified from this hydrolysate, demonstrates that a histidine residue(s) is the site of phosphorylation.

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Authors and Affiliations

  1. Department of Microbiology, Arizona State University, 85287, Tempe, Arizona, USA

    Hsiu-Ping P. Lin &  Henry C. Reeves

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  1. Hsiu-Ping P. Lin
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  2. Henry C. Reeves
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Lin, HP.P., Reeves, H.C. Properties of phosphorylated NADP+-specific glutamate dehydrogenase fromEscherichia coli . Current Microbiology 24, 73–79 (1992). https://doi.org/10.1007/BF01570901

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