Structure-function relationships in TPN-dependent isocitrate dehydrogenase. I. Electron paramagnetic resonance studies of the interaction of enzyme-bound Mn(II) with substrates, cofactors, and substrate analogues.
Electron paramagnetic resonance (EPR) spectra were obtained for various isocitrate dehydrogenase-Mn(II) complexes. The qualitative effects of the binding of substrates, nucleotides, and substrate analogues on the isotropic character of the electronic environment of enzyme-bound Mn(II) were subsequently investigated. The addition of isocitrate produces a markedly anisotropic spectrum whereas alpha-ketoglutarate does not alter the spectrum of enzyme-Mn(II) substantially. This suggests direct coordination of isocitrate to the Mn(II) but perphaps a different mode of binding for alpha-ketoglutarate. Other studies demonstrated mutually exclusive binding relationships between TPN and TPNH, between Mn-isocitrate and TPNH, and between HCO3-(CO2) and formate or thiocyanate. Indirect evidence supporting CO2 rather than HCO3-as the actual reactive species which binds to the enzyme in the reductive carboxylation reaction is presented on the basis of the results of the formate and thiocyanate studies. From the EPR results recorded for ternary, quaternary, and quinary enzyme-substrate complexes, correlations between the appearance of fine structure signals and the binding of individual substrates and/or nucleotides are found, and tentative assignments of such signals are made on this basis. Additional studies were conducted to determine binding constants for Mg(II) Co(II), and Co-isocitrate, and a comparison was made with kinetically determined binding constants.
D000818:Animals; D001665:Binding Sites; D003035:Cobalt / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology; D009249:NADP; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013552:Swine
D003035:Cobalt; D008345:Manganese; D009249:NADP; D007521:Isocitrate Dehydrogenase