el Kebbaj M S, Latruffe N
Arch Biochem Biophys. 1986 Feb 1;244(2):662-70. doi: 10.1016/0003-9861(86)90634-x.
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.
已通过动力学方法研究了磷脂与D-β-羟丁酸脱氢酶（BDH，一种需要卵磷脂的酶）之间的相互作用。磷脂对BDH的再激活过程遵循二级机制，这表明：（1）至少2摩尔卵磷脂对于该酶的再激活至关重要；（2）该酶含有两个对卵磷脂有依赖性的结合位点。再激活时间进程的图形表示显示出一个潜伏期，当磷脂量增加时该潜伏期会缩短。对再激活动力学数据进行Scatchard图处理表明存在两类磷脂结合位点，分别与四个和两个卵磷脂分子的结合相关，表现出高亲和力和低亲和力。温度对BDH活性以及用N，N'-二环己基碳二亚胺（一种特异性羧基试剂）或苯乙二醛（一种特异性精氨酸试剂）对脱辅酶进行失活的影响在22-24℃出现转折，表明在该温度附近酶活性位点的结构发生了轻微变化。此外，根据磷脂的性质，酶动力学参数的变化与磷脂的性质和流动性状态相关的构象变化一致。然而，表观NAD +结合常数并不取决于磷脂的流动性。
