The existence of a monomer-dimer equilibrium with lipoamide dehydrogenase is demonstrated. The equilibrium can be shifted to the monomer side at low ionic strength and low pH by removing the phosphate ions by extensive dialysis. At low ionic strength, I : 0.01 and 0.02, the enzyme precipitates while aggregation takes place. This aggregation seems to be due to changes in the activity coefficient of the enzyme. High phosphate concentrations, NADI and high temperatures favor association. Also bringing the enzyme in a more polar environment causes dissociation. Dioxan and 2-chloroethanol are used to decrease the dielectric constant of the buffer solution. Inactivation and dissociation of the enzyme is time- dependent in these solutions. High concentrations of dioxan and 2-chloroethanol cause denaturation and precipitation of the enzyme. High phosphate concentrations stimulate the denaturation and precipitation of the enzyme in dioxan and 2-chloroethanol.
Dissociation of the enzyme is accompanied by loss in activity and decrease in apparent α-helix content. ORD and CD data show this decrease, however the possibility that this decrease is due to changes in shape and size of the protein molecule cannot be excluded. Fluorescence and CD experiments show that upon dissociation an amino acid, a tryptophan residue, moves to a more polar environment. Also by treating the enzyme with dioxan a tryptophan residue is pertubed.
Dissociation of the enzyme can also be achieved by treating the enzyme with sodium dodecylsulfate. Hydrophobic and ionic interactions are observed. Binding to the hydrophobic sites, by sodium dodecylsulfate or Tween 80, has no influence on the lipoate activity and on absorption spectrum of the enzyme in the visible- region. Binding to the ionic sites causes loss in lipoate activity and affects the absorption spectrum. From the dependency on the pH and the ionic strength it is concluded that a group of the kind BH +
= B + H +
with a pK value around 6.6 is involved. At high SDS concentrations the binding of FAD to the enzyme is weakened and upon standing for long times the flavin dissociated off.
Dimerization of the enzyme is favored by NAD +
. Binding of NAD +
to the enzyme yields a difference spectrum. From these spectral titration curves two pairs of NAD +
-binding sites are calculated, the binding site with the highest affinity, K diss
= 35 μM is assigned to the regulatory site while the binding site with K diss
= 90-110 μM is assigned to the catalytic site. Upon NAD +
binding to the regulatory site one proton per FAD is liberated. Comparision of the pH activity curves with computer models shows that the activating effect of NAD +
in the lipoate activity can be explained by a shift in pK value of a group from pH 6.4-6.3 to 5.0-4.9 upon NAD +
binding. Together with observations in the literature these results suggest that the pK value of a SH-group is shifted to lower pH upon NAD +
binding. This SH-group is suggested to be functional in the S -
state in the active center.