Cooperativity in lipid activation of 3-hydroxybutyrate dehydrogenase: role of lecithin as an essential allosteric activator.
Cooperativity in lipid activation of 3-hydroxybutyrate dehydrogenase: role of lecithin as an essential allosteric activator.
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3-羟基丁酸脱氢酶脂质激活的协同作用：卵磷脂作为重要变构激活剂的作用。
DOI：
10.1021/bi00433a040
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Fleischer,S
中科院分区：
文献类型：
--
作者：
Cortese,JD;McIntyre,JO;Duncan,TM;Fleischer,S
范德比尔特大学，分子生物学系，纳什维尔，田纳西州37235接收于1988年8月11日;修订的Mandarin pt接收于1988年12月2日摘要：3-羟基丁酸脱氢酶（BDH）是一种需要卵磷脂的线粒体酶，以NAD（H）作为辅酶催化3-羟基丁酸和乙酰乙酸的相互转化。纯化的酶没有脂质（即脱辅基脱氢酶或apoBDH）可以重新激活与可溶性卵磷脂或插入到磷脂囊泡含有卵磷脂。已经提出了两种不同的模型来解释S形脂质激活曲线。对于这两种模型，假定BDH的活化需要每个功能单元结合两个卵磷脂分子。由短链（可溶性）卵磷脂的solubleenzyme（二聚体形式）的激活是一致的模型，其中卵磷脂结合是非合作的，而膜结合酶（tetramericform）的激活表明卵磷脂结合位点之间的协同性。提出了一个新的综合模型，其中卵磷脂被认为是一个必不可少的变构激活剂，改变酶的构象状态之间的平衡。共振能量转移数据，反映NADH结合到膜boundandsoluble apoBDH，是一致的，这样的卵磷脂诱导的构象变化。对于由双层和可溶性卵磷脂激活的BDH，NADH与BDH结合的表观解离常数分别为~ 10 µ和~ 37 µ。最大荧光共振能量转移（AFmM）随卵磷脂在双层膜中摩尔分数的增加而增加。最大的变化发生在摩尔分数0和0.13之间，从而与酶的功能相关。在没有卵磷脂的情况下，基本上没有观察到NADH的结合。变构模型调和了可溶性磷脂和双层磷脂对BDH的明显不同的激活，并且可以解释先前观察到的用于酶与可溶性磷脂的激活的结合位点之间缺乏协同性[Córtese，J.D.，维达尔，J.C.，丘吉尔，P.，麦金太尔，J. O.，&弗莱舍，S.（1982）Biochemistry 21，3899-3908]。卵磷脂对BDH的非协同和协同活化都是更一般的变构模型的限制性情况。1.30，（R）-3-羟基丁酸：NAD+氧化还原酶，BDH] 1具有S形形状。已经提出了两种模型来解释S形激活（Córtese等人，1982; Sandermann等人，1986年）。关于卵磷脂结合，具有两个相同的非相互作用卵磷脂结合位点的非合作模型与可溶性卵磷脂的活化一致（Córtese等人，1982年）。一个模型，
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235 Received August 11, 1988; Revised Manuscript Received December 2, 1988 abstract: 3-Hydroxybutyrate dehydrogenase (BDH) is a lecithin-requiring mitochondrial enzyme which catalyzes the interconversion of 3-hydroxybutyrate and acetoacetate with NAD (H) as coenzyme. Thepurified enzyme devoid of lipid (ie, the apodehydrogenase or apoBDH) can be reactivated with soluble lecithin or by insertion into phospholipid vesicles containing lecithin. Twodifferent models have been proposed to explain the sigmoidal lipid activation curves. For both models, activation of BDH is assumed to require the binding of two lecithin molecules per functional unit. Activation of solubleenzyme (dimeric form) by short-chain (soluble) lecithin is consistent with a model in which lecithin binding is noncooperative, whereas activation of the membrane-bound enzyme (tetramericform) indicates cooperativity between the lecithin binding sites. A new comprehensive model is presentedin which lecithin is considered to be an essential allosteric activator that shifts the equilibrium between conformational states of the enzyme. Resonance energy transfer data, reflecting NADH binding to membrane-boundand soluble apoBDH, are consistent with such a lecithin-induced conformational change. Apparent dissociationconstants for binding of NADH to BDH are~ 10 µ and~ 37 µ for BDH activated by bilayer and soluble lecithin, respectively. The maximal fluorescence resonance energy transfer (AFmM) increases with highermole fraction of lecithin in the bilayer. The largest changes occur between mole fractions 0 and 0.13, thereby correlating with enzymic function. Essentially no binding ofNADH is observed in the absence of lecithin. The allosteric model reconciles the apparentlydistinct activation of BDH by soluble and bilayer phospholipid and can account for the lack of cooperativity between binding sites observed previously for the activation of the enzyme with soluble phospholipid [Córtese, J. D., Vidal, J. C., Churchill, P., McIntyre, J. O., & Fleischer, S.(1982) Biochemistry 21, 3899-3908], Both noncooperative and cooperative activation ofBDH by lecithin are limiting cases of the more general allosteric model.Tjjpid activation curves for 3-hydroxybutyrate dehydrogenase [EC 1.1. 1.30,(R)-3-hydroxybutyrate: NAD+ oxidoreductase, BDH] 1 have a sigmoidal shape. Two models have been pro-posed to explain sigmoidal activation (Córtese et al., 1982; Sandermann et al., 1986). With regard to lecithin binding, a noncooperative model with two identical noninteracting lecithin binding sites was consistent with the activation by soluble lecithins (Córtese et al., 1982). A model involving