Reactivation of D-beta-hydroxybutyrate dehydrogenase with short-chain lecithins: stoichiometry and kinetic mechanism.
D-beta-Hydroxybutyrate dehydrogenase (BDH), purified as soluble, lipid-free apoenzyme (inactive) from either beef heart or rat liver mitochondria, can be reactivated by short-chain lecithins in the monomeric state. The enzyme was reactivated with dihexanoyl- [PC(6:0)], diheptanoyl- [PC(7:0)], and dioctanoyllecithins [PC(8:0)]. The titration curves of enzyme activity as a function of the phospholipid concentration are consistent with a model in which the enzyme contains two identical, noninteracting lecithin binding sites. The simultaneous occupation of these sites (via an equilibrium random mechanism) is required to activate the apoenzyme. Similar results were obtained with both rat liver and beef heart apoenzymes. The maximal velocities obtained with the different lecithins were similar [110-140 mumol of NAD+ reduced min-1 (mg of protein)-1]. The KL values (the apparent dissociation constants of the lecithin-site complexes) were 1.2 X 10(-4) M [PC(8:0)], 1.5 X 10(-3) M [PC(7:0)], and 4.5 X 10(-3) M [PC(6:0)] at 37 degrees C. This was confirmed by using phospholipase A2 to compete with the dehydrogenase for the lecithin monomers. Comparison of the delta G degrees values for complex formation with the different lecithins shows an average contribution of approximately 2.4 kJ/mol (0.9RT) per CH2 group. The interaction of the apolar moiety of lecithin with the protein seems to be essential for effective binding of phosphatidylcholine to apoBDH. The delta G degrees values, when combined with the estimated delta H degrees values, suggest that the binding of lecithin to the apoenzyme is approximately 60% enthalpy and approximately 40% entropy driven.
D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D006884:Hydroxybutyrate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D010713:Phosphatidylcholines / Q000494:pharmacology*; D051381:Rats
D010713:Phosphatidylcholines; D006884:Hydroxybutyrate Dehydrogenase