Interactions between apo-(d-β-hydroxybutyrate dehydrogenase) and phospholipids studied by intrinsic and extrinsic fluorescence

Abstract

Interactions of D-beta-hydroxybutyrate dehydrogenase with phospholipids were investigated by both intrinsic- and extrinsic-fluorescence approaches. The intrinsic fluorescence, mainly caused by tryptophan residues, increased upon re-activation in the presence of phospholipids bearing a positive charge, i.e. phosphatidylcholine, but decreased in the presence of non-re-activating phospholipids with a negative charge. This indicates either that the environment of tryptophan residues is affected by charges rather than by hydrophobic chains of phospholipids, or that the enzyme undergoes different conformational changes depending on the nature of the phospholipids. On the other hand, the graph of the temperature-dependence of the fluorescence intensities of the enzyme embedded in dimyristoylphosphatidylcholine liposomes exhibits a break around 21 degrees C. This indicates either that at least one tryptophan residue is closely in contact with the hydrophobic chains of phospholipids or that there is a change in the environment of tryptophan residues owing to the physical state of the phospholipids. The addition of D-beta-hydroxybutyrate apo-dehydrogenase to phospholipid liposomes containing diphenylhexatriene (a fluorescent probe) increased the diphenylhexatriene fluorescence polarization. Moreover, there was a partial fluorescence energy transfer from tryptophan to diphenylhexatriene. These results strongly favour the possibility that there is a portion of the enzyme polypeptide chain inserted into the phospholipid hydrophobic region. All these results demonstrate that D-beta-hydroxybutyrate apo-dehydrogenase interacts with both polar and hydrophobic parts of phospholipids and leads to small, but essential, conformational changes of the enzyme.

Coenzyme binding of 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme

“…Binding of nucleotide by the apodehydrogenase was suggested by some of our previous chemical modification studies which showed that high concentrations of NAD(H) reduced the rate of derivatization by cyclohexanedione for both the apodehydrogenase and the reconstituted enzyme ). Studies of the quenching of intrinsic fluorescence of the rat liver apodehydrogenase by NADH also suggested binding of the nucleotide but in a concentration range similar to that observed with the enzyme-phospholipid complex (El Kebbaj et al, 1986). However, the direct binding studies reported here demonstrate that the binding of nucleotide to the apodehydrogenase is much weaker than binding of nucleotide to the active enzyme.…”

On the Origin of ATP Synthesis in Cancer

“…Moreover, ketone bodies enhance the metabolic efficiency of normal host cells, but are growth inhibitory or even toxic to many tumor cells (Clarke et al, 2012;Veech, 2004;Cahill and Veech, 2003;Bartmann et al, 2018;Skinner et al, 2009;Fine et al, 2009;Poff et al, 2017;Magee et al, 1979;Hagihara et al, 2020;Ji et al, 2020). Abnormalities in cardiolipin and other phospholipids in the inner mitochondrial membranes would prevent tumor cells from using ketone bodies for ATP synthesis (El Kebbaj et al, 1986;Kiebish et al, 2008Kiebish et al, , 2009. Through their anti-angiogenic, anti-inflammatory, and pro-apoptotic actions, calorie restriction and KD-R can normalize the tumor microenvironment (Zhou et al, 2007;Marsh et al, 2008;Mukherjee et al, 2002Mukherjee et al, , 2004Mukherjee et al, , 2019Mulrooney et al, 2011;Shelton et al, 2010a;Urits et al, 2012).…”