A carboxylate oxygen of the substrate bridges the magnesium ions at the active site of enolase: Structure of the yeast enzyme complexed with the equilibrium mixture of 2-phosphoglycerate and phosphoenolpyruvate at 1.8 angstrom resolution
A carboxylate oxygen of the substrate bridges the magnesium ions at the active site of enolase: Structure of the yeast enzyme complexed with the equilibrium mixture of 2-phosphoglycerate and phosphoenolpyruvate at 1.8 angstrom resolution
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DOI：
10.1021/bi952859c
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发表时间：
1996-04-09
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：
Larsen, TM;Wedekind, JE;Reed, GH
酵母烯醇化酶与底物2-磷酸-D-甘油酸（2-PGA）和产物磷酸烯醇丙酮酸（P-烯醇丙酮酸）的平衡混合物已在pH 8.0的聚乙二醇（PEG）溶液中结晶。晶体属于空间群C2，晶胞尺寸为a 121.9埃，B = 73.2埃，c 93.9埃，β 93.3度。平衡混合物的晶体和膦酰基乙酰异羟肟酸（PhAH）的烯醇化酶复合物的晶体是同晶型的，并且前者复合物的结构从烯醇化酶-（Mg 2+）2-PhAH的坐标解出[Wedekind，J，E.，波伊纳河R.，里德湾，加-地H、& Rayment，I.（1994）Biochemistry 33，9333-9342]。对于1.8埃分辨率的所有记录数据（92%完成），当前晶体学R因子为17.7%。电子密度图对于两种镁离子的位置和配位以及对于底物/产物结合的立体化学是明确的。两种镁离子都与底物/产物的官能团络合。较高亲和力的Mg 2+与Asp 246、Glu 295和Asp 320的羧酸酯侧链（底物/产物的羧酸酯氧和水分子）配位。底物/产物的羧酸根氧之一也与较低亲和力的Mg 2 +-配位，从而形成μ-羧酸根桥。第二Mg 2+的其他配体是底物/产物的磷酰基氧、两个水分子以及来自活性位点环的Ser 39的羰基和γ-氧。两种镁离子与羧酸根基团的复杂配位表明两种金属离子都参与稳定碳负离子（酸-羧酸根）中间体中的负电荷。Lys 345的ε-氨基被定位为在正向反应中充当碱基，而Glu 211的羧酸酯侧链被定位为与2-PGA的3-OH相互作用。结构提供了一个坦率的看法烯醇化酶的催化机制。
The equilibrium mixture of yeast enolase with substrate, 2-phospho-D-glycerate (2-PGA), and product, phosphoenolpyruvate (P-enolpyruvate), has been crystallized from solutions of poly(ethylene glycol) (PEG) at pH 8.0. Crystals belong to the space group C2 and have unit cell dimensions a 121.9 Angstrom, b = 73.2 Angstrom, c 93.9 Angstrom, and beta 93.3 degrees. The crystals have one dimer per asymmetric unit, Crystals of the equilibrium mixture and of the enolase complex of phosphonoacetohydroxamate (PhAH) are isomorphous, and the structure of the former complex was solved from the coordinates of enolase-(Mg2+)2-PhAH [Wedekind, J, E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994) Biochemistry 33, 9333-9342]. The current crystallographic R-factor is 17.7% for all recorded data (92% complete) to 1.8 Angstrom resolution. The electron density map is unambiguous with respect to the positions and liganding of both magnesium ions and with respect to the stereochemistry of substrate/product binding. Both magnesium ions are complexed to functional groups of the substrate/product. The higher affinity Mg2+ coordinates to the carboxylate side chains of Asp 246, Glu 295, and Asp 320, both carboxylate oxygens of the substrate/ product, and a water molecule. One of the carboxylate oxygens of the substrate/product also coordinates to the lower affinity Mg2+-thus forming a mu-carboxylato bridge. The other ligands of the second Mg2+ are a phosphoryl oxygen of the substrate/product, two water molecules, and the carbonyl and gamma-oxygens of Ser 39 from the active site loop. The intricate coordination of both magnesium ions to the carboxylate group suggests that both metal ions participate in stabilizing negative charge in the carbanion (aci-carboxylate) intermediate. The epsilon-amino group of Lys 345 is positioned to serve as the base in the forward reaction whereas the carboxylate side chain of Glu 211 is positioned to interact with the 3-OH of 2-PGA. The structure provides a candid view of the catalytic machinery of enolase.