Distances among coenzyme and metal sites of NADP+-dependent isocitrate dehydrogenase using resonance energy transfer.
When the substrate isocitrate-Mn2+ is present, the fluorescent nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) reacts irreversibly with pig heart NADP+-specific isocitrate dehydrogenase at the coenzyme binding site on one subunit of the dimeric enzyme [Bailey, J. M., & Colman, R. F. (1985) Biochemistry 24, 5367-5377]. The modified enzyme, which retains partial activity, binds 1 mol of NADPH or 1 mol of the coenzyme analogue, reduced thionicotinamide adenine dinucleotide phosphate (TNADPH), per dimer. TNADPH quenches the fluorescence of enzyme-bound 2-BDB-T epsilon A-2',5'-DP with an efficiency of energy transfer of 9.8%. From this value and the spectral properties of the donor and acceptor chromophores, a distance of 32 A was calculated as the average distance between coenzyme sites on the two subunits. Isocitrate dehydrogenase activity requires a divalent metal ion, such as Mn2+, Co2+, or Ni2+. Co2+ and Ni2+ have absorption spectra that overlap the emission spectra of enzyme-bound 2-BDB-T epsilon A-2',5'-DP. In the presence of isocitrate, each of these two metal ions quenches the fluorescence of the enzyme-bound reagent with an efficiency of energy transfer of 28-29%. From this value and the spectral characteristics of the energy donor and acceptors, an average distance of 8.0 A was estimated between the metal-isocitrate site and the labeled coenzyme site. These distances have provided constraints in formulating a model of the spatial arrangement of active-site ligands on isocitrate dehydrogenase.
Bailey
J M
JM
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Colman
R F
RF
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004735:Energy Transfer; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D009249:NADP / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013552:Swine
D000345:Affinity Labels; D008345:Manganese; D009249:NADP; D000244:Adenosine Diphosphate; C047570:2-((4-bromo-2,3-dioxobutyl)thio)-1,N(6)-ethenoadenosine 2',5'-bisphosphate; D007521:Isocitrate Dehydrogenase