The structure of Yeast Enolase at 2.25-Å Resolution: An 8-fold β + α-Barrel with a Novel β β α α (β α)6 Topology

Abstract

The three-dimensional structure of yeast enolase has been determined by the multiple isomorphous replacement method followed by the solvent flattening technique. A polypeptide model, corresponding with the known amino acid sequence, has been fitted to the electron density map. Crystallographic restrained least-squares refinement of the model without solvent gave R = 20.0% for 6-2.25-A resolution with good geometry. A model with 182 water molecules and 1 sulfate which is still being refined has presently R = 17.0%. The molecule is a dimer with subunits related by 2-fold crystallographic symmetry. The subunit has dimensions 60 × 55 × 45 A and is built from two domains. The smaller N-terminal domain has an α + β structure based on a three-stranded antiparallel meander and four helices. The main domain is an 8-fold β + α-barrel. The enolase barrel is, however, different from the triose phosphate isomerase barrel; its topology is β β α α (β α)6 rather than (β α)8 as found in triose phosphate isomerase. The inner β-barrel is not entirely parallel, the second strand is antiparallel to the other strands, and the direction of the first helix is also reversed with respect to the other helices. This supports the hypothesis that some enzymes evolved independently producing the stable structure of β α barrels with either enolase or triose phosphate isomerase topology. The active site of enolase is located at the carboxylic end of the barrel. A fragment of the N-terminal domain and two long loops protruding from the barrel domain form a wide crevice leading to the active site region. Asp246, Glu295, and Asp320 are the ligands of the conformational cation. Other residues in the active site region are Glu168, Asp321, Lys345, and Lys396.