Larsen T M, Wedekind J E, Rayment I, Reed G H
Institute for Enzyme Research, University of Wisconsin-Madison, 53705, USA.
Biochemistry. 1996 Apr 9;35(14):4349-58. doi: 10.1021/bi952859c.
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 A, b = 73.2 A, c = 93.9 A, 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 A 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.
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）的平衡混合物已从pH 8.0的聚乙二醇（PEG）溶液中结晶出来。晶体属于空间群C2，晶胞尺寸为a = 121.9 Å，b = 73.2 Å，c = 93.9 Å，β = 93.3°。每个不对称单元中有一个二聚体。平衡混合物的晶体与膦酰基乙酰羟肟酸（PhAH）的烯醇化酶复合物晶体是同晶型的，前者复合物的结构是根据烯醇化酶-(Mg2+)2-PhAH的坐标解析得到的[韦德金德，J. E.，波伊纳，R. R.，里德，G. H.，& 雷门特，I.（1994年）《生物化学》33，9333 - 9342]。对于所有记录数据（92%完整），至1.8 Å分辨率，当前晶体学R因子为17.7%。电子密度图对于两个镁离子的位置和配位以及底物/产物结合的立体化学是明确的。两个镁离子都与底物/产物的官能团络合。亲和力较高的Mg2+与天冬氨酸246、谷氨酸295和天冬氨酸320的羧酸盐侧链、底物/产物的两个羧酸盐氧原子以及一个水分子配位。底物/产物的一个羧酸盐氧原子也与亲和力较低的Mg2+配位——从而形成一个μ-羧基桥。第二个Mg2+的其他配体是底物/产物的一个磷酰氧原子、两个水分子以及来自活性位点环的丝氨酸39的羰基和γ-氧原子。两个镁离子与羧酸盐基团的复杂配位表明两个金属离子都参与稳定碳负离子（烯醇羧酸盐）中间体中的负电荷。赖氨酸345的ε-氨基在正向反应中作为碱，而谷氨酸211的羧酸盐侧链则与2-PGA的3-OH相互作用。该结构为烯醇化酶的催化机制提供了直观的视角。
