Mechanism of enolase: the crystal structure of enolase-magnesium-2-phosphoglycerate/phosphoenolpyruvate complex at 2.2-.ANG. resolution

Abstract

Enolase in the presence of Mg2+ catalyzes the elimination of H2O from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP) and the reverse reaction, the hydration of PEP to PGA. The structure of the ternary complex yeast enolase-Mg2(+)-PGA/PEP has been determined by X-ray diffraction and refined by crystallographic restrained least-squares to an R = 16.9% for those data with I/sigma (I) greater than or equal to 2 to 2.2-A resolution with a good geometry of the model. The structure indicates the substrate molecule in the active site has its hydroxyl group coordinated to the Mg2+ ion. The carboxylic group interacts with the side chains of His373 and Lys396. The phosphate group is H-bonded to the guanidinium group of Arg374. A water molecule H-bonded to the carboxylic groups of Glu168 and Glu211 is located at a 2.6-A distance from carbon-2 of the substrate in the direction of its proton. We propose that this cluster functions as the base abstracting the proton in the catalytic process. The proton is probably transferred, first to the water molecule, then to Glu168, and further to the substrate hydroxyl to form a water molecule. Some analogy is apparent between the initial stages of the enolase reverse reaction, the hydration of PEP, and the proteolytic mechanism of the metallohydrolases carboxypeptidase A and thermolysin. The substrate/product binding is accompanied by large movements of loops Ser36-His43 and Ser158-Gly162. The role of these conformational changes is not clear at this time.

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

“…These results were later proved in Lebioda and Reed laboratories when the crystalline structures of yeast enolase and neuron-specific human enolase were established [2,45,46]. Selective mutagenesis of yeast enolase and its recent crystallographic studies revealed that the basic amino-acids Lys345, Lys396, His159, His373, and Arg374 in the catalytic center are important in the mechanism of the reversible transition of 2-PGA to PEP.…”

“…During gluconeogenesis (anabolic pathway) the same enzyme as a phosphopyruvate hydratase catalyses the reverse reaction hydration of PEP to 2-PGA. Magnesium ions are a natural cofactor of enolase, essential in maintaining active conformation of the catalytic domain and important in the enzymatic reaction mechanism [2]. Enolase is one of most abundantly expressed cytosolic proteins in many organisms.…”

Posttranscriptional and Posttranslational Control of Enolase Expression in the Facultative Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L

“…indicate positions of conservative amino acid replacement. Residues that are thought to participate in substrate binding (E'73, H382, R383, and K405) are indicated with a closed circle ( 0 ) (Lebioda and Stec, 1991). Conserved amino acid residues (D25', E302, and D3") that are likely to serve as ligands for conformational Mg2+ binding are indicated with plus signs (+).…”

Function ofDunaliella salina(Dunaliellaceae) enolase and its expression during stress

“…The crystal structures of enolases from Escherichia coli, Homarus gammarus and Saccharomyces cerevisiae have been determined at high resolution (Stec & Lebioda, 1990;Lebioda & Stec, 1991;Duquerroy et al, 1995;Zhang et al, 1997;Chai et al, 2004). The above authors have reported that eukaryotic enolases are dimeric and some bacterial enolases are octameric; furthermore, these enolases have very similar amino acid sequences and three-dimensional structures.…”