MECHANISM OF ENOLASE - THE CRYSTAL-STRUCTURE OF ASYMMETRIC DIMER ENOLASE-2-PHOSPHO-D-GLYCERATE ENOLASE-PHOSPHOENOLPYRUVATE AT 2.0 ANGSTROM RESOLUTION/

Enolase, a glycolytic enzyme that catalyzes the dehydration of 2-phosp ho-D-glycerate (PGA) to form phosphoenolpyruvate (PEP), is a homodimer in all eukaryotes and many prokaryotes. Here, we report the crystal s tructure of a complex between yeast enolase and an equilibrium mixture of PGA and PEP. The structure has been refined using 29 854 reflectio ns with an F/sigma(F) of greater than or equal to 3 to an R of 0.137 w ith average deviations of bond lengths and bond angles from ideal valu es of 0.013 Angstrom and 3.1 degrees respectively. In this structure, the dimer constitutes the crystallographic asymmetric unit. The two su bunits are similar, and their superposition gives a rms distance betwe en C-alpha atoms of 0.91 Angstrom. The exceptions to this are the cata lytic loop Val153-Phe169 where the atomic positions in the two subunit s differ by up to 4 Angstrom 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 su bstrate/product molecule; in the other it is separated by water molecu les. 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. Th e electron density present in the active site suggests that in the act ive site with the direct ligand-His159 hydrogen bond PGA is predominan tly bound while in the active site where water molecules separate His1 59 from the ligand the binding of PEP dominates. The structure indicat es that the water molecule hydrating carbon-3 of PEP in the PEP-->PGA reaction is activated by the carboxylates of Glu168 and Glu211. The cr ystals are unique because they have resolved two intermediates on the opposite sides of the transition state.