8-(4-Bromo-2,3-dioxobutylthio)NAD: a new affinity label for NAD-specific isocitrate dehydrogenase.
8-(4-Bromo-2,3-dioxobutylthio)nicotinamide adenine dinucleotide (8-BDB-TNAD), a new reactive NAD analog, was synthesized by coupling 8-thio-AMP with NMN, followed by condensation with 1,4-dibromobutanedione. Incubation of 160 microM 8-BDB-TNAD with the allosteric pig heart NAD-dependent isocitrate dehydrogenase causes time-dependent inactivation to a limit of 25% residual activity concomitant with incorporation of approximately 1 mol reagent/mol average subunit. In addition to binding sites for NAD and NADH, this enzyme has been shown to have regulatory sites for NADPH and for ADP (R. S. Ehrlich and R. F. Colman 1982, J. Biol. Chem. 257, 4769-4774). Marked protection against enzyme inactivation by 8-BDB-TNAD and incorporation is provided by the regulatory nucleotides NADPH or ADP, while NAD and NADH are less effective. The rate constant for inactivation shows a nonlinear dependence on 8-BDB-TNAD concentration which can be ascribed to reversible formation of an enzyme-reagent complex (KI = 83 microM) prior to an irreversible reaction (kmax = 0.0625 min-1). Analysis of the kinetic properties and binding characteristics of modified enzyme indicates that this enzyme retains the ability to bind ADP, but does not bind NADPH. Thus, 8-BDB-TNAD reacts at or near the allosteric NADPH site of pig heart NAD-dependent isocitrate dehydrogenase.
Kumar
A
A
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Colman
R F
RF
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013552:Swine
D000345:Affinity Labels; D009243:NAD; C085867:8-(4-bromo-2,3-dioxobutylthio)NAD; D007521:Isocitrate Dehydrogenase