title: Distance between the substrate and regulatory reduced coenzyme binding sites of bovine liver glutamate dehydrogenase by resonance energy transfer.
abstract: Bovine liver glutamate dehydrogenase is known to bind reduced coenzyme at two sites/subunit, one catalytic and one regulatory; ADP competes for the latter site. The enzyme is here shown to be catalytically active with the thionicotinamide analogue of NADPH [( S]NADPH). For native enzyme, ultrafiltration studies revealed that [S]NADPH reversibly occupies about two sites/enzyme subunit in the absence of other ligands; by the addition of ADP, [S]NADPH binding can be limited to one molecule/subunit. The enzyme is irreversibly inactivated by reaction with 4-(iodoacetamido)salicylic acid (ISA) at lysine126 within the 2-oxoglutarate binding site [Holbrook, J.J., Roberts, P.A. & Wallis, R.B. (1973) Biochem. J. 133, 165-171]. ISA-modified enzyme binds 1 molecule [S]NADPH/subunit in the absence of ADP, suggesting that reaction at the substrate site blocks binding at the catalytic, but not at the regulatory site. The fluorescence spectrum of ISA-modified enzyme overlaps the absorption spectrum of [S]NADPH allowing a distance measurement between these sites by resonance energy transfer. [S]NADPH quenches the emission of ISA-modified enzyme, yielding 3.2 nm as the average distance between sites. ADP competes for the [S]NADPH site but does not affect the fluorescence of ISA-modified enzyme, indicating that [S]NADPH quenching is attributable to energy transfer rather than to a conformational change. The 3.2 nm thus represents the distance between the 2-oxoglutarate and reduced coenzyme regulatory sites of glutamate dehydrogenase.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
authors.forename: R H
authors.initials: RH
authors.lastname: Lark
authors.forename: R F
authors.initials: RF
authors.lastname: Colman
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive; D002417:Cattle; D004735:Energy Transfer; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D007527:Isoenzymes / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D009249:NADP / Q000032:analysis; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D007527:Isoenzymes; D009249:NADP; D000244:Adenosine Diphosphate; D005969:Glutamate Dehydrogenase