Interactions of Neurospora crassa plasma membrane H+-ATPase with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.
Interactions of Neurospora crassa plasma membrane H+-ATPase with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.
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粗糙脉孢菌质膜 H-ATP 酶与 N-(乙氧基羰基)-2-乙氧基-1,2-二氢喹啉的相互作用。
DOI：
10.1021/bi00362a013
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Scarborough,GA
中科院分区：
文献类型：
--
作者：
Addison,R;Scarborough,GA
摘要：羧基活化剂A-(乙氧羰基)-2-乙氧基-1，2-二氢喹啉(EEDQ)以至少三种不同的方式与脉孢霉质膜H~+-ATPase相互作用。该试剂不可逆地抑制ATP水解酶，在几种浓度的EEDQ下呈假一级动力学，这些数据的适当转换表明，1molEEDQ使1molH+-ATPase失活。抑制可能涉及激活ATPase羧基，然后由ATPase多肽链中邻近的亲核官能团进行亲核攻击，从而导致分子内交联。在钒酸盐存在的情况下，镁ATP可以保护酶不受EEDQ抑制，钒酸盐是一种配体的组合，它可以将H+-ATPase“锁定”在一种可能类似于酶的磷酸化和去磷酸化反应的过渡态的构象中，但不受底物类似物镁ADP的保护，这与依赖EEDQ的抑制性分子内交联反应中涉及的一个或两个残基通常参与ATP的-y-磷酸基转移，或与之接近的观点是一致的。在EEDQ依赖的反应中，ATPase也可以被外源亲核分子[14C]甘氨酸乙酯标记，而在MgATP和钒的存在下，标记作用减弱。然而，[~(14)C]甘氨酸乙酯标记的ATPase的肽图表明，该标记与EEDQ抑制反应没有任何简单的联系。在第三种类型的相互作用中，EEDQ介导ATPase单体与其他膜蛋白(可能是另一种ATPase单体)的特定交联，导致形成表观分子量约为26万的产物。这个反应发生得比抑制反应慢得多，因此可能也没有直接关系。丝状真菌粗面脉孢子菌质膜中的主要ATP水解酶是一个电生质子泵(Scarborough，1976,1980)，能够产生超过200 mV的跨膜电位差(Slayman等人，1973)。该酶的水解部分的分子质量约为105000道尔顿(Dame&Scarborough，1980，Addison&Scaror-
Revised Manuscript Received March 12, 1986 abstract: The carboxyl group activating reagent A-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline (EEDQ) interacts with the Neurospora plasma membrane H+-ATPase in at least three different ways. This reagent irreversibly inhibits ATP hydrolysis with kinetics that are pseudo-first-order atseveral concentrations of EEDQ, and an appropriate transform of these data suggests that 1 mol of EEDQinactivates 1 mol of the H+-ATPase. Inhibition probably involves activation of an ATPase carboxyl group followed by a nucleophilic attack by a vicinal nucleophilic functional group in the ATPase polypeptide chain, resulting in an intra-molecular cross-link. The enzyme is protected against EEDQ inhibition by MgATP in thepresence of vanadate, a combination of ligands that has previously been shown to “lock” the H+-ATPase in a conformation that presumably resembles the transition states of the enzyme phosphorylation and dephosphorylation reactions, but is not protected by the substrate analogue MgADP, which is consistent with the notion that one or both of the residues involved in the EEDQ-dependent inhibitory intramolecular cross-linking reaction normally participate in the transfer of the-y-phosphoryl group of ATP, or are near those that do. The ATPase is also labeled by the exogenous nucleophile [14C] glycine ethyl ester in an EEDQ-dependent reaction, and the labeling is diminished in the presence of MgATP plus vanadate. However, peptide maps of [l4C] glycine ethyl ester labeled ATPase demonstrate that the labeling is not related to the EEDQ inhibition reaction in any simple way. In a third type of interaction, EEDQ mediates the specific cross-linking of ATPase monomers with some other membrane protein, possibly another ATPase monomer, leading to the formation of a product with an apparent molecular weight of about 260 000. This reaction occurs substantially more slowly than the inhibition reaction and is thus presumably not directly related either. e principal ATP hydrolyzing enzyme in the plasma membrane of the filamentous fungus Neurospora crassa is an electrogenic proton pump (Scarborough, 1976, 1980) capable of generating a transmembrane electrical potential difference in excess of 200 mV (Slayman et al., 1973). The hydrolytic moiety of this enzyme has a molecular mass of about 105 000 daltons (Dame & Scarborough, 1980, Addison & Scarbor-