Conformations of the coenzymes and the allosteric activator, ADP, bound to NAD(+)-dependent isocitrate dehydrogenase from pig heart.
NAD(+)-dependent isocitrate dehydrogenase from pig heart is an allosteric enzyme that is activated by ADP and is inhibited by NADPH in the presence of NADH. Transferred nuclear Overhauser effect measurements, made at a range of times to ensure that observed effects are due to direct dipole-dipole transfer and not to spin diffusion, were used to determine the conformations of pyridine nucleotide coenzymes and of the allosteric effector ADP. For NAD+, significant effects were observed on the N2 proton (on the nicotinamide ring) when the N1' proton (on the nicotinamide ribose) was saturated and on the N6 proton when the N2' proton was saturated, indicating that the conformation of the nicotinamide-ribose moiety is anti. The anti conformation is expected because of the stereospecificity of NAD(+)-dependent isocitrate dehydrogenase and is the same as for NADP(+)-dependent isocitrate dehydrogenase. For the adenosine moiety of NAD+, the predominant nuclear Overhauser effect on the A8 proton is found when the A2' proton is saturated. This result implies that the adenine-ribose bond is anti with respect to the ribose. Previous kinetic and binding studies of ADP activation have shown an influence of divalent metal ions. The conformation of bound ADP, in the presence of Mg2+ and/or Ca2+, is found to be anti about the adenine-ribose bond. The 3'H-8H distance increases when Ca2+ is added to the Mg-ADP-enzyme complex. Changes in the 4'H-1'H distance upon addition of isocitrate are indicative of interactions between the ADP activator site and the isocitrate site.(ABSTRACT TRUNCATED AT 250 WORDS)
Ehrlich
R S
RS
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Colman
R F
RF
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003067:Coenzymes / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006321:Heart / Q000187:drug effects; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology*; D009249:NADP / Q000494:pharmacology; D011487:Protein Conformation; D013552:Swine
D003067:Coenzymes; D009243:NAD; D009249:NADP; D000244:Adenosine Diphosphate; D007521:Isocitrate Dehydrogenase; D008274:Magnesium; D002118:Calcium