Inactivation of NAD-dependent isocitrate dehydrogenase by affinity labeling of the allosteric ADP site by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate.
A new reactive ADP analogue has been synthesized: 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate (2-BDB-TADP). Reaction of ADP with m-chloroperoxybenzoic acid gave ADP 1-oxide, which was treated with NaOH, followed by reaction with carbon disulfide to yield 2-thioadenosine 5'-diphosphate. The final product was synthesized by condensation of 2-thioadenosine 5'-diphosphate with 1,4-dibromobutanedione. Reaction of pig heart NAD-specific isocitrate dehydrogenase with this nucleotide analogue (0.4 mM) causes a time-dependent loss of activity to a limiting value of 75% inactivation. The rate constant for inactivation exhibits a nonlinear dependence on the concentration of 2-BDB-TADP, with kmax = 0.021 min-1 and KI = 0.067 mM. Complete protection against inactivation by 0.2 mM 2-BDB-TADP is provided by ADP + Mn2+, but not by Mn2+ alone, isocitrate, alpha-ketoglutarate, or NAD. Incorporation of 2-BDB-TADP is proportional to the extent of inactivation, reaching 1 mol of reagent/mol of enzyme subunit when the enzyme is maximally inactivated. However, when inactivation is totally prevented by incubation with 2-BDB-TADP in the presence of ADP and Mn2+, 0.5 mol of reagent/mol of subunit is still incorporated, suggesting that inactivation may be attributed to 0.5 mol of reagent/mol of average subunit. In the native enzyme, the Km for total isocitrate is 1.8 mM and is decreased 6-fold to 0.3 mM in the presence of 1 mM ADP, whereas in the modified enzyme, with 25% residual activity, the Km for total isocitrate is about the same in the absence (2.0 mM) or presence (1.8 mM) of ADP. These results indicate that 2-BDB-TADP acts as an affinity label of the ADP allosteric site of NAD-dependent isocitrate dehydrogenase.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008433:Mathematics; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D013552:Swine; D013873:Thionucleotides / Q000378:metabolism*
D000345:Affinity Labels; D007656:Ketoglutaric Acids; D013873:Thionucleotides; D009243:NAD; D000249:Adenosine Monophosphate; D008345:Manganese; D000244:Adenosine Diphosphate; C042298:6-((4-bromo-2,3-dioxobutyl)thio)-6-deaminoadenosine 5'-diphosphate; D007521:Isocitrate Dehydrogenase