Enolase
Created by Michael Pokrass
Enolase (pdb = 1IYX) from Enterococcus hirae is an enzyme that participates in the glycolytic pathway by binding to 2-phosphoglyceric acid (PGA) and converting it to phosphoenol pyruvate (PEP). Enolases are present in the cells of virtually all organisms and possess significant structural similarities across these organisms. Enolases from different species may resemble one another structurally due to their functional similarities in the glycolytic pathways of cells (Hosaka, et al., 822). ExPASy is a bioinformatics tool that is useful in determining qualities of proteins such as isoelectric points and molecular weights. ExPASy also serves as a site that can direct investigators to other resources and databases to elucidate structural and functional features of proteins. According to ExPASy the molecular weight of Enolase from E. hirae is 46,411Da and the isoelectric point is 4.58 (Artimo, et al., 1).
Glycolysis refers to a set of sequential biochemical reactions that occur in almost all cells in which glucose or other sugars are broken down to ultimately obtain energy for a cell or organism (Garrett & Grisham, 577-580). Enolase catalyzes the dehydration of PGA to PEP late in the second phase of the glycolytic pathway. One proposed reaction mechanism is an anti β-elimination in which the ε-amino group of Lys-339 abstracts a proton from the central carbon of PGA, followed by the acid-catalyzed elimination of the hydroxyl group at C3. The acid catalysis is achieved with the γ-carboxyl group of Glu-204 (Hosaka, et al., 817-818). The ε-amino of Lys-390, along with a Mg2+ ion, participates in the mechanism by stabilizing the negatively charged carboxyl group of PGA (Reed, et al., 737). Although the ΔG of this reaction is relatively small at only 1.8kJ/mol, the phosphoenol product has a substantially greater free energy of hydrolysis, which makes PEP suitable for ATP synthesis in the subsequent reaction of the pathway (Garrett & Grisham, 592-594).
Enolase consists of two identical subunits of 431 residues each. The primary structure of enolase is divided into two domains. The N-terminal domain consists of the first 133 residues and contains an antiparallel β-sheet of three strands and four alpha helices (Hosaka, et al., 820-821). The C-terminal domain consists of residues 134-431 and contains the active site and a mixed α/β-barrel, which displays an unusual folding pattern that gives ββαα(βα)6 connectivity (Reed, et al., 737). These two domains are connected through a loop region called L4 that refers to residues 132-140 (Hosaka, et al. 820-821). Each monomer of enolase has a secondary structure composed of approximately 40% helices made up of 18 helices totaling of 177 residues and 17% beta sheets made up by 22 strands totaling 75 residues (Kabsch & Sandler, 2577-637). There are three loop regions that are important to catalytic function and are more flexible than other regions of the protein because they shift towards the active site after binding to the substrate occurs. These loop regions are L1 (residues 38-45), L2 (residues 152-149) and L3 (residues 244-265) (Hosaka, et al., 820-821).
The α/β-barrel architecture in the C-terminal domain creates a deep crater that ends at the catalytic site of the enzyme. Eight b-strands form the β-sheet that serves as the cylindrical interior of the active site. Eight a-helices surround the β-sheet. The 11th sheet and helix are also involved in non-covalent interactions that stabilize the plane in which two enolase subunits interact to dimerize. The residues at the C-terminals of the β-strands in the barrel are involved in the enzymatic mechanism and are hydrophilic, while the residues before these in the strands tend to be hydrophobic (Kühnel & Luisi, 583, 588).
Under physiological conditions enolase is present in the cytosol as a homodimer (Reed, et al., 737). The carbonyl carbon (Co) of Arg-396 facilitates the dimerization of enolase monomers by establishing hydrogen bonds with the amide groups of Arg-399 and Ile-400 (Hosaka, et al., 822). Each monomeric subunit of enolase must bind two divalent metal cations in order for the enzyme to perform its catalytic function. Mg2+ is normally the metal cofactor that enolase binds. The first Mg2+ ion binds with greater affinity in the active site of the enzyme and causes a conformational change that allows the subsequent binding of substrate. The second Mg2+ ion binds with lesser affinity and will only bind after the substrate has entered the active site. The binding of the second Mg2+ ion causes the reversible dehydration of PGA to occur (Hosaka, et al., 817). It has been observed that binding of Mg2+ and other divalent cations encourages dimerization of enolase, which suggests that the cations stabilize the dimer structure (Kühnel & Luisi, 588). Monovalent cations such as Li+ and Na+ have been observed to partially inhibit the enzymatic activity of enolase. Mg2+ competes with Li+ to bind and the binding of Li+ decreases the rate at which PEP is produced (Kornblatt & Musil, 301).
PSI-BLAST stands for position-specific-iterated basic local alignment search tool. PSI-BLAST is a tool that compares proteins in terms of their primary structures and assigns them an “E-value” that is reflective of their sequence homology. As E-values approach zero, sequence homology increases. The Dali server is a tool that finds proteins that have similarities in their folds. It does this by comparing the distances between molecules in the proteins. Tertiary structural similarity is reported as a “Z-score”. Greater values of Z-scores are indicative of greater similarity between a given pair of proteins compared with a pair of proteins with lower Z-scores. Enolases from Escherichia coli and Enterococcus hirae have an E-value of 0.0 and a Z-score of 63.9. According to the Dali server, the two enolases are 65% identical and PSI-BLAST indicates that the enzymes have a maximum identity of 65% (Altschul, et al., 3389-3402; Holm, W545-549). Because of these similarities, E. coli enolase (pdb = 1E9I) was chosen as a comparison protein to E. hirae enolase.
The primary, secondary, and tertiary structures of E. coli enolase greatly resemble those of E. hirae enolase. The sequence of E. coli enolase is also 431 residues and divided into an N-terminal domain and a C-terminal domain (Kabsch & Sandler, 2577-637). The C-terminal domain contains the ββαα(βα)6 barrel that leads to the active site. The secondary structure of E. Coli enolase is 41% helical with 19 helices made up of 181 residues and 18% β-sheet with 24 strands made up of 78 residues. E. hirae enolase has one fewer helix and two fewer β-strands; however, these differences in secondary structure are not part of the secondary or tertiary structure of the active site or the L1, L2, and L3 regions that are involved in catalytic activity (Kühnel & Luisi, 583-587). In both enolases the dimer interface is predominantly composed of hydrophobic residues; however, the specific residues that contribute to the stabilization of the dimer interface are slightly different. The Co of Arg-396 in E. hirae enolase interacts with the amide N of Arg-399 and Ile-400. The amide N of Arg-396 forms hydrogen bonds with Asp-398 and the Co of Ser-14 (Hosaka, et al., 822). The Co of Arg-398 in E. coli enolase interacts with the N atoms of Arg-401 and Val-402. The amide N of Arg-398 forms hydrogen bonds with the Co of Ser-14 and the amide N of Ser-399 interacts with the carboxyl group of Asp-400 (Kühnel & Luisi, 588). Although these differences are minor, this is not unexpected because the structures of enolases have been highly conserved throughout evolution to preserve function. The different interactions between residues may have an effect on protein features such as thermostability or dimerization affinity.