Kinetic aspects of the role of phospholipids in D-beta-hydroxybutyrate dehydrogenase activity.
Interactions of phospholipids with D-beta-hydroxybutyrate dehydrogenase (BDH), a lecithin-requiring enzyme, have been studied by a kinetic approach. The process of reactivation of BDH by phospholipids, which follows a second-order mechanism, reveals that (1) at least 2 mol of lecithins is essential for the reactivation of the enzyme, and (2) the enzyme contains two dependent binding sites for lecithins. The graphic representation of the time course of reactivation shows a latent phase which decreases when there is an increase in the amount of phospholipids. A Scatchard plot treatment of the reactivation kinetic data reveals the presence of two classes of phospholipid binding sites, which exhibit high and low affinities related to the binding of four and two lecithin molecules, respectively. The effect of temperature on BDH activity and on the inactivation of the apoenzyme with N,N'-dicyclohexylcarbodiimide (a specific carboxyl reagent) or with phenylglyoxal (a specific arginine reagent) shows a break at 22-24 degrees C, indicating a slight structural change in the enzyme-active site around this temperature. In addition, the variations in enzyme kinetic parameters, according to the nature of phospholipids, are in agreement with conformational changes related to the nature and to the fluidity state of phospholipids. However, the apparent NAD+ binding constant does not depend on the phospholipid's fluidity.
D000818:Animals; D001051:Apoenzymes / Q000032:analysis; D001665:Binding Sites; D004789:Enzyme Activation; D006884:Hydroxybutyrate Dehydrogenase / Q000032:analysis*; D066298:In Vitro Techniques; D007700:Kinetics; D008563:Membrane Lipids / Q000032:analysis; D009243:NAD / Q000378:metabolism; D010713:Phosphatidylcholines / Q000502:physiology; D010743:Phospholipids / Q000032:analysis / Q000502:physiology*; D051381:Rats; D013696:Temperature
D001051:Apoenzymes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010743:Phospholipids; D009243:NAD; D006884:Hydroxybutyrate Dehydrogenase