Binding of small molecules to lipoamide dehydrogenase

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

Identification of the thiol residues involved in modifications of pig heart lipoamide dehydrogenase by cupric ion and by iodoacetamide

The thiol residues involved in two previously described modifications of heart lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) have been identified by comparison of peptide maps of unmodified and modified enzymes. Two thiols and one methionine react when native enzyme is alkylated in concentrated iodoacetamide, with the accompanying loss of enzymatic activity and an NAD-binding site. NAD protects the more slowly reacting thiol from akylation, and the NAD-protected enzyme is active and retains its NAD-binding site. Loss of the binding site, and loss of activity are associated with the alkylation of two neutral thiol peptides which may represent alternative versions of a single thiol region in the enzyme. Treatment of native enzyme with cupric ion results in the rapid oxidation of two thiols to a disulfide bond and loss of NADH-lipoamide reductase activity. We have determined that thiol residues in the cationic peptide and one of the anionic peptides are involved in the disulfide bond formed by cupric ion. Since the cationic peptide contains two histidyl residues, it is proposed that it is the initial site of binding of cupric ion, prior to the oxidation of the thiol residues to a disulfide bond.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22231/1/0000665.pd

Cryptic proteolytic activity of dihydrolipoamide dehydrogenase

The mitochondrial enzyme, dihydrolipoamide dehydrogenase (DLD), is essential for energy metabolism across eukaryotes. Here, conditions known to destabilize the DLD homodimer enabled the mouse, pig, or human enzyme to function as a protease. A catalytic dyad (S456–E431) buried at the homodimer interface was identified. Serine protease inhibitors and an S456A or an E431A point mutation abolished the proteolytic activity, whereas other point mutations at the homodimer interface domain enhanced the proteolytic activity, causing partial or complete loss of DLD activity. In humans, mutations in the DLD homodimer interface have been linked to an atypical form of DLD deficiency. These findings reveal a previously unrecognized mechanism by which certain DLD mutations can simultaneously induce the loss of a primary metabolic activity and the gain of a moonlighting proteolytic activity. The latter could contribute to the metabolic derangement associated with DLD deficiency and represent a target for therapies of this condition