Brewer J M, Carreira L A, Collins K M, Duvall M C, Cohen C, DerVartanian D V
J Inorg Biochem. 1983 Nov;19(3):255-67. doi: 10.1016/0162-0134(83)85030-2.
Measurements of binding of certain divalent cations to yeast apoenolase were made using a pH-meter, chromatography, a divalent cation electrode, and ultrafiltration. The binding of the activating metal ions Mg2+ and Co2+ and the nonactivator Ca2+ were studied as functions of the presence or absence of substrate/product, phosphate, and fluoride or level of Tb3+. The data suggest phosphate and fluoride increase Mg2+ binding but not Ca2+ binding. Substrate/product appears to increase Ca2+ binding as well as that of Mg2+ and Co2+. In the presence of substrate, Co2+ binding was 5-6 mol/mol dimer. In the absence of substrate/product, Tb3+ reduced Co2+ binding from 4 mol/mol to 2. These data are interpreted in terms of binding to "conformational," "catalytic" (substrate/product dependent), and "inhibitory" sites. Measurements of Tb3+ fluorescence quenching by Co2+ suggested that the distance between "conformational" sites on the two subunits was large, while the distance between "conformational" and "inhibitory" sites was ca. 17 +/- 4 A. Potentiometric titrations of apoenzyme with Ca2+ and Mg2+ showed that the metal ions produced the same proton release in the presence or absence of substrate/product. If phosphate and fluoride were present, then more protons were released if Ca2+ was the titrant rather than Mg2+, suggesting a difference in ionization state in the complex with the activating metal. Electron paramagnetic resonance studies of Co2+ binding to the various sites in the enzyme are presented. The Co2+ bound to all three sites appears to be high spin, consistent with a preponderance of oxyligands in an octahedral environment. Substrate, citrate, and a strongly binding substrate analogue strongly enhance the hyperfine structure of conformational Co2+. This is interpreted as the result of a change in interaction of an axial ligand to conformational Co2+ produced by carbon-3 of substrate or analogue.
利用pH计、色谱法、二价阳离子电极和超滤法对某些二价阳离子与酵母脱辅基烯醇酶的结合进行了测量。研究了激活金属离子Mg2+和Co2+以及非激活剂Ca2+的结合情况，考察了底物/产物、磷酸盐、氟化物的存在与否或Tb3+水平对其的影响。数据表明，磷酸盐和氟化物会增加Mg2+的结合，但不会增加Ca2+的结合。底物/产物似乎会增加Ca2+以及Mg2+和Co2+的结合。在有底物存在的情况下，Co2+的结合量为5 - 6摩尔/摩尔二聚体。在没有底物/产物的情况下，Tb3+会将Co2+的结合量从4摩尔/摩尔降低到2摩尔/摩尔。这些数据是根据与“构象”、“催化”（依赖于底物/产物）和“抑制”位点的结合来解释的。Co2+对Tb3+荧光猝灭的测量表明，两个亚基上“构象”位点之间的距离较大，而“构象”和“抑制”位点之间的距离约为17±4埃。用Ca2+和Mg2+对脱辅基酶进行电位滴定表明，无论有无底物/产物，金属离子产生的质子释放量相同。如果存在磷酸盐和氟化物，那么以Ca2+作为滴定剂时比以Mg2+作为滴定剂释放的质子更多，这表明与激活金属形成的复合物中离子化状态存在差异。本文介绍了Co2+与酶中各个位点结合的电子顺磁共振研究。结合到所有三个位点的Co2+似乎都是高自旋的，这与八面体环境中氧配体占优势一致。底物、柠檬酸盐和一种强结合底物类似物会强烈增强构象Co2+的超精细结构。这被解释为底物或类似物的碳-3产生的轴向配体与构象Co2+相互作用发生变化的结果。
