title: Chelation of serine 39 to Mg2+ latches a gate at the active site of enolase: structure of the bis(Mg2+) complex of yeast enolase and the intermediate analog phosphonoacetohydroxamate at 2.1-A resolution.
abstract: The structure of a new crystal form of enolase from bakers' yeast has been solved to 2.1-A resolution. Crystals were grown from poly(ethylene glycol) and KCl at pH 8.2 in the presence of Mg2+ and a reaction intermediate analog, phosphonoacetohydroxamate (PhAH). Crystals belong to space group C2; have unit cell dimensions a = 123.5 A, b = 73.9 A, and c = 94.8 A with beta = 93.3 degrees; and contain one dimer per asymmetric unit. The structure was solved by molecular replacement from the X-ray coordinates of apoenolase [Stec, B., & Lebioda, L. (1990) J. Mol. Biol. 211, 235-248]. Both essential divalent metal ions are observed to be complexed with the inhibitor. The two Mg2+ ions are 4.05 A apart and are bridged by a mu-oxyl ligand from the carbonyl moiety of PhAH. The "high-affinity" Mg2+ coordinates to the carboxylate side chains of Asp 246, Glu 295, and Asp 320, one water molecule, and the hydroxamate and carbonyl oxygens of PhAH. The second Mg2+ coordinates to a phosphonyl oxygen, two water molecules, and the mu-bridge carbonyl oxygen of PhAH. Coordination schemes with respect to PhAH and water ligands are fully consistent with those of the Mn2+ complexes determined spectroscopically [Poyner, R.R., & Reed, G. H. (1992) Biochemistry 31, 7166-7173]. Remaining ligands for the second Mg2+ are the carbonyl oxygen and gamma-oxygen of Ser 39. Chelation of this Ser residue to Mg2+ effectively "latches" a flexible loop extending from Gly 37 through His 43 and closes off the entrance to the active site. The position of the second Mg2+ in the active site provides new insight into the stereochemistry of substrate binding.
authors.affiliation: Institute for Enzyme Research, Graduate School, University of Wisconsin, Madison 53705.
authors.forename: J E
authors.initials: JE
authors.lastname: Wedekind
authors.forename: R R
authors.initials: RR
authors.lastname: Poyner
authors.forename: G H
authors.initials: GH
authors.lastname: Reed
authors.forename: I
authors.initials: I
authors.lastname: Rayment
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002614:Chelating Agents; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006877:Hydroxamic Acids / Q000378:metabolism*; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism*; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012694:Serine; D013816:Thermodynamics
chemical_list: D002614:Chelating Agents; D006877:Hydroxamic Acids; D046911:Macromolecular Substances; D012694:Serine; C087412:phosphonoacetohydroxamate; D010751:Phosphopyruvate Hydratase; D008274:Magnesium; D010746:Phosphonoacetic Acid