| Abstract
| - The equilibrium mixture of yeast enolase with substrate,2-phospho-d-glycerate (2-PGA), andproduct, phosphoenolpyruvate (P-enolpyruvate), has been crystallizedfrom solutions of poly(ethyleneglycol) (PEG) at pH 8.0. Crystals belong to the space groupC2 and have unit cell dimensions a =121.9Å, b = 73.2 Å, c = 93.9 Å, and β =93.3°. The crystals have one dimer per asymmetric unit.Crystalsof the equilibrium mixture and of the enolase complex ofphosphonoacetohydroxamate (PhAH) areisomorphous, and the structure of the former complex was solved fromthe coordinates of enolase−(Mg2+)2−PhAH [Wedekind, J. E., Poyner, R.R., Reed, G. H., & Rayment, I. (1994) Biochemistry33,9333−9342]. The current crystallographic R-factor is17.7% for all recorded data (92% complete) to 1.8Å resolution. The electron density map is unambiguous withrespect to the positions and liganding ofboth magnesium ions and with respect to the stereochemistry ofsubstrate/product binding. Both magnesiumions are complexed to functional groups of the substrate/product.The higher affinity Mg2+ coordinatesto the carboxylate side chains of Asp 246, Glu 295, and Asp 320, bothcarboxylate oxygens of the substrate/product, and a water molecule. One of the carboxylate oxygens ofthe substrate/product also coordinatesto the lower affinity Mg2+thus forming aμ-carboxylato bridge. The other ligands of the secondMg2+are a phosphoryl oxygen of the substrate/product, two water molecules,and the carbonyl and γ -oxygensof Ser 39 from the active site loop. The intricate coordination ofboth magnesium ions to the carboxylategroup suggests that both metal ions participate in stabilizing negativecharge in the carbanion(aci-carboxylate) intermediate. The ε-amino group ofLys 345 is positioned to serve as the base in theforward reaction whereas the carboxylate side chain of Glu 211 ispositioned to interact with the 3-OHof 2-PGA. The structure provides a candid view of the catalyticmachinery of enolase.
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