STRUCTURE OF THE BIS DIVALENT-CATION COMPLEX WITH PHOSPHONOACETOHYDROXAMATE AT THE ACTIVE-SITE OF ENOLASE
STRUCTURE OF THE BIS DIVALENT-CATION COMPLEX WITH PHOSPHONOACETOHYDROXAMATE AT THE ACTIVE-SITE OF ENOLASE
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DOI：
10.1021/bi00146a020
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发表时间：
1992-08-11
期刊：
影响因子：
2.9
通讯作者：
REED, GH
中科院分区：
文献类型：
--
作者：
POYNER, RR;REED, GH
膦酰乙酰异羟肟酸盐（PhAH）是烯醇化酶的紧密结合（K（i）= 15 pM）抑制剂，据信其模拟反应中中间体碳负离子的酸-羧酸盐形式[安德森，V.E.，韦斯，P. M.，& Cleland，W. W.（1984）Biochemistry 23，2779]。用Mn ~（2+）的电子顺磁共振（EPR）谱研究了PhAH与面包酵母烯醇化酶活性位点上两种二价阳离子的相互作用。含有两个Mn ~（2+）结合在活性位点的烯醇酶-PhAH复合物的EPR谱含有多个精细结构跃迁，每个跃迁具有45-G Mn-55超精细间距，这是Mn ~（2+）的自旋交换耦合对的特征。 磁稀释的配合物，通过制备特定的Mg 2 +/Mn 2+杂化配合物，通过操纵的二价金属物种的添加顺序。因此，通过向烯醇化酶和PhAH的溶液中加入1当量的Mn 2+，然后加入1当量的Mg 2+，将Mn 2+置于较高亲和力位点。颠倒Mg 2+和Mn 2+的添加顺序将Mn 2+置于较低亲和力位点。区域特异性O-17-标记形式的PhAH的制备，并确定从存在或不存在的O-17 superhyperfine耦合在EPR信号中的两个金属离子的网站上的功能基团的结合的PhAH到Mn 2+。异羟肟酸氧是Mn 2+在较高亲和力位点的配体，膦酸酯氧是Mn 2+在较低亲和力位点的配体，羰基氧是两个金属离子的μ-O桥。结合的抑制剂的双核螯合物结构表明亲电作用的两个二价阳离子在稳定的酸-羧酸盐形式的中间体碳负离子以及亲电作用的高亲和力的金属离子在增强的离去基团的能力的3-OH的2-磷酸-D-甘油酸。
Phosphonoacetohydroxamate (PhAH) is a tight-binding (K(i) = 15 pM) inhibitor of enolase that is believed to mimic the aci-carboxylate form of the intermediate carbanion in the reaction [Anderson, V. E., Weiss, P. M., & Cleland, W. W. (1984) Biochemistry 23, 2779]. Electron paramagnetic resonance (EPR) spectroscopy of Mn2+ has been used to map sites of interaction of PhAH with the two divalent cations at the active site of enolase from bakers' yeast. EPR spectra of enolase-PhAH complexes containing two Mn2+ bound at the active site contain multiple fine structure transitions each with a 45-G Mn-55 hyperfine spacing that is a characteristic of spin exchange coupled pairs of Mn2+. Magnetically dilute complexes were obtained by preparation of specific Mg2+/Mn2+ hybrid complexes by manipulating the order of addition of the divalent metal species. Thus, Mn2+ was placed in the higher affinity site by addition of 1 equiv of Mn2+ to a solution of enolase and PhAH, followed by addition of 1 equiv of Mg2+. Reversing the order of addition of Mg2+ and Mn2+ placed Mn2+ in the lower affinity site. Regiospecifically O-17-labeled forms of PhAH were prepared, and the binding of the functional groups on PhAH to Mn2+ at the two metal ion sites was determined from the presence or absence of O-17 superhyperfine coupling in the EPR signals. The hydroxamate oxygen is a ligand of Mn2+ at the higher affinity site, a phosphonate oxygen is a ligand of Mn2+ at the lower affinity site, and the carbonyl oxygen is a mu-O bridge of the two metal ions. The binuclear chelate structure of the bound inhibitor suggests electrophilic roles for both divalent cations in stabilization of the aci-carboxylate form of the intermediate carbanion as well as an electrophilic role of the high-affinity metal ion in augmenting the leaving group ability of the 3-OH of 2-phospho-D-glycerate.