Identification of cysteine and lysine residues present at the active site of beef liver glutamate dehydrogenase by o-phthalaldehyde.
Beef liver glutamate dehydrogenase (GDH) is inactivated by the bifunctional reagent, o-phthalaldehyde. The initial rate of inactivation follows pseudo first-order kinetics. The reaction of the enzyme with o-phthalaldehyde results in isoindole derivative formation which is characterized by typical fluorescence emission and excitation maximum at 410 nm and 337 nm, respectively. The inactivation of GDH by o-phthalaldehyde is partially prevented by alpha-ketoglutaric acid, whereas NADH does not provide any protection. This clearly indicates that cysteine and lysine residues are located near the alpha-ketoglutaric acid binding center. The dissociation constant of 2.2 mM was obtained for enzyme-alpha-ketoglutaric acid complex. Stoichiometry of o-phthalaldehyde binding with glutamate dehydrogenase showed that the formation of approximately one isoindole derivative per subunit of glutamate dehydrogenase is accompanied by complete loss of activity.
Pandey
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Department of Chemistry, Indian Institute of Technology Kanpur, India.
Sheikh
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Katiyar
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D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003545:Cysteine / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008239:Lysine / Q000737:chemistry*; D008623:Mercaptoethanol / Q000494:pharmacology; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D009764:o-Phthalaldehyde / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007211:Indoles; D007656:Ketoglutaric Acids; D009243:NAD; D002731:4-Chloromercuribenzenesulfonate; D008623:Mercaptoethanol; D009764:o-Phthalaldehyde; D005969:Glutamate Dehydrogenase; D008239:Lysine; D003545:Cysteine