Binding of nicotinamide nucleotides to dihydrolipoamide dehydrogenase measured with spin-labeled analogs

Binding of NAD and NADH to dihydrolipoamide dehydrogenase fromEscherichia coli and from pig heart was measured using the spin-labeled analogsN⁶-(2,2,6,6-tetramethylpiperidine-4-yl-1-oxyl)-NAD and -NADH. A decrease in the peak amplitudes of the respective EPR spectra results after adding enzyme to the cofactor analogs. With the bacterial enzyme normal hyperbolic saturation behavior with the NAD analog and one binding site per subunit (K_s=0.51 mM) are observed, while the NADH analog reveals a sigmoidal binding characteristic. A high-affinity and a low-affinity site (K_s=0.087 and 0.33 mM) are found for binding of the NAD analog to the pig heart enzyme and only one type of binding site is observed for the NADH analog (K_s=22 µM).