Conformational studies on lipoamide dehydrogenase from pig heart. 4. The binding of NAD + to non-equivalent sites.

Abstract

NAD+ forms two pairs of spectrally distinguishable complexes upon binding to oxidized lipoamide dehydrogenase. The shape and magnitude of the spectra is dependent on temperature and pH. The flavin‐absorption band at 450 nm is not shifted uniformly. Intrinsic dissociation constants only could be calculated at 430 nm. In 30 mM phosphate buffer pH 7.2 at 25 °C K1d= 50–60 μM, while at 0 °C K1d= 40 μM for the high‐affinity pair; for the low‐affinity pair these values are 200–250 μM and 110–130 μM respectively. The high‐affinity pair is also present at pH 5.6 at I= 0.1 and I= 0.2, the values at 25 °C are respectively 50 μM and 35 μM. Computer simulation suggests that the activating effect of NAD+ on the NADH : lipoate reductase activity can be explained by assuming that binding of NAD+ shifts the pK‐value of a protonated group at the active centre from 6.2 to 4.9–5.0. This suggestion is supported by the observation that binding of NAD+ at the high‐affinity site is accompanied by liberation of 0.9 ± 0.07 equiv. H+/mol flavin. From titration data it was calculated that for NAD+ binding K1d= 65 μM. The results indicate that binding of NAD+ at the high‐affinity site, even at partial saturation, shifts the monomer‐dimer equilibrium towards the dimer. Furthermore the difference in reduction state upon reducing the enzyme with NADH at 25 °C and 0 °C is not due to differences in affinity for NAD+, but to conformational changes of the protein around the flavin moiety. It has to be considered that the two active centres of the dimer are not completely independent.