Mechanism of Enolase:  The Crystal Structure of Asymmetric Dimer Enolase−2-Phospho-d-glycerate/Enolase−Phosphoenolpyruvate at 2.0 Å Resolution^,

Abstract

Enolase, a glycolytic enzyme that catalyzes the dehydration of 2-phospho-d-glycerate (PGA) to form phosphoenolpyruvate (PEP), is a homodimer in all eukaryotes and many prokaryotes. Here, we report the crystal structure of a complex between yeast enolase and an equilibrium mixture of PGA and PEP. The structure has been refined using 29 854 reflections with an F/sigma(F) of >/=3 to an R of 0.137 with average deviations of bond lengths and bond angles from ideal values of 0.013 A and 3.1 degrees , respectively. In this structure, the dimer constitutes the crystallographic asymmetric unit. The two subunits are similar, and their superposition gives a rms distance between Calpha atoms of 0.91 A. The exceptions to this are the catalytic loop Val153-Phe169 where the atomic positions in the two subunits differ by up to 4 A and the loop Ser250-Gln277, which follows the catalytic loop Val153-Phe169. In the first subunit, the imidazole side chain of His159 is in contact with the phosphate group of the substrate/product molecule; in the other it is separated by water molecules. A series of hydrogen bonds leading to a neighboring enolase dimer can be identified as being responsible for ordering and stabilization of the conformationally different subunits in the crystal lattice. The electron density present in the active site suggests that in the active site with the direct ligand-His159 hydrogen bond PGA is predominantly bound while in the active site where water molecules separate His159 from the ligand the binding of PEP dominates. The structure indicates that the water molecule hydrating carbon-3 of PEP in the PEP --> PGA reaction is activated by the carboxylates of Glu168 and Glu211. The crystals are unique because they have resolved two intermediates on the opposite sides of the transition state.

Lateral transfer at the gene and subgenic levels in the evolution of eukaryotic enolase

“…Mapping the insertion region to the crystal structure of yeast enolase (25)(26) shows that the insertions are located in a loop, and not on the surface where enolase monomers interact in forming homodimers (25)(26), suggesting that this region is amenable to alterations in size and shape. Indeed, insertions are found at the same locations in diplomonads, in some genes from red algae, and in one gene from a green alga, further implying that these regions, like others in enolase (27), are prone to insertions and deletions.…”

Crystal structure of the Escherichia coli RNA degradosome component enolase

“…18 One subunit was present in the closed conformation, while the second was observed in a``loose'' intermediate conformation.…”

“…Compared to animal enolases, this loop is on average four residues shorter in prokaryotic enolases and two residues longer in plant enolases. 18 This loop moves during the closure of the active site upon substrate binding. The root-mean-square ®t of C a atom positions for the core regions, excluding loops, is 0.71 A Ê and 0.76 A Ê for matching the E. coli enzyme with yeast (3enl) and lobster (1pdy) enolases, respectively.…”

“…17,23 An alternative proposal suggests that Glu211 and Glu168 share a proton, which then catalyzes the abstraction of the hydroxy group. 18 In yeast and lobster enolase, it has been observed that the binding of substrate causes major conformational changes in the enzyme that closes the substrate into the active site. In yeast enolase, the N-terminal domain contributes a lonḡ exible loop that closes on the active site by binding a magnesium ion via residue Ser39 (corresponding to E. coli residue 41) and a second loop (153-169) containing His159 (E. coli: His158) moves closer to the active site.…”

Stopped‐flow studies of the reaction of d‐tartronate semialdehyde‐2‐phosphate with human neuronal enolase and yeast enolase 1

“…There is considerable crystallographic evidence [8,[22][23][24]] that binding of substrate/analogues at the active sites of both enzymes is accompanied by movement of polypeptide loops that close around the active sites. (Since crystals are routinely prepared with saturating levels of all ligands, it is not presently possible to say whether substrate binding alone -no catalytic Mg 2+ bound -produces any loop(s) movement, but it is plausible that some movement occurs [7].)…”