Pyruvate kinase (PBD ID: 1A3W) from Saccharomyces cerevisiae
Created by: Ramya Ravi
Pyruvate kinase (PBD ID: 1A3W) is a transferase enzyme in the metabolism pathway found in Saccharomyces cerevisiae (S. cerevisiae). Pyruvate kinase converts phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP, catalyzing the final step in glycolysis by transferring the phosphate group from PEP to ADP. This enzyme is a key control point in metabolism that is allosterically regulated by fructose-1,6-bisphosphate (FBP). In humans, there are four isozymes (L, R, M1 and M2) that depend on tissue type and physiological conditions. This enzyme not only plays a key role in metabolism, but also in cell proliferation (1). Research is being done to see if this enzyme can be used to target malignant cells, since they would have higher rates of aerobic glycolysis and more M2 isozymes (1,2,3).
Pyruvate kinase extracted from S. cerevisiae was crystallized through vapor diffusion in hanging drops under the condition of a pH of 6.5. Structural data was determined by X-ray diffraction crystallography. The entire protein was crystalized and represented in the published structure, with only a few, negligible number of residues missing at the N terminus (1,4).
The Expasy database reports that pyruvate kinase has a molecular weight of 109071.94 Da and an isoelectric point of 7.77 (5). Made up of two identical chains (A and B), pyruvate kinase is a homodimer that often forms homotetramers (2,3). Pyruvate kinase is made up of 1,000 total residues, with 500 residues and four domains for each of the two subunits (1,4).
The N Domain is a short α-helical stretch at the N terminus. The A domain, formed by residues 19-88 and 189-360 that fold together into parallel α/β barrel motifs with 2 additional helices, is the main catalyzing site. The B domain is the capping domain that is formed by residues 89-188, which form 9 stranded β-barrels that create the cap over active site. The C domain, made up of α/β open sheets from residues 361-500, is the regulatory domain located at the C terminus and is allosterically regulated by FBP (1).
The primary structure of pyruvate kinase contains 1,000 total residues. The secondary structure is 38% helical (21 helices, 90 residues), 16% β-sheets (27 strands, 82 residues), with approximately three 3/10 helices and the rest are random coils. The alpha helices allow for hydrophobic collapse and structural stabilization by having the hydrophobic amino acids on the interior of the core and the acidic and basic residues on the outside. The resulting hydrogen bonds between helices also help stabilize this structure. Electrostatic interactions form between the positively charged potassium ion (K+), manganese (II) ion (Mn2+), Arg-49 and Lys-269 and the negatively charged phosphates of 2-phosphoglycolic acid (PG) or PEP. The tertiary structure is determined by the structures of the four domains. Quaternary structures are formed with the dimers and possible tetramers of pyruvate kinase through hydrogen bonding across subunits and salt bridges, especially between Arg-369 and Asp-366, which help stabilize the interaction (1).
Pyruvate kinase transfers the phosphate group from PEP to ADP when allosterically activated by FBP, forming the second ATP in glycolysis. This step is the control point before starting either anaerobic fermentation or oxidative phosphorylation. The allosteric regulation of pyruvate kinase helps maintain levels of ATP and glycolytic intermediates. The conformation change allows for the binding of PEP to the active site in the A domain. The affinity of PEP to this domain is increased when FBP is bound to the C (regulatory) domain 40 Å away. The four ligands of pyruvate kinase are FBP, PG, K+ and Mn2+(1).
The carboxylate side-chain oxygens of Glu-242 and Asp-266 help form the octahedral coordination sphere to bind Mn2+. The two stretches of amino acids between residues 51-55 and 83-92 are highly conserved sequences that form the site to bind K+. Arg-49 and Lys-269 have positive side chains that combine with K+ and Mn2+ to form a positive electrostatic pocket to attract the phosphate in PG, which activates the phophoryl transfer process by making the phosphorous atom more susceptible to the nucleophillic attack. The 1' phosphate group of FBP, the allosteric activator, forms an electrostatic interaction with Arg-459 and the 6'phosphate group of FBP forms H-bonds with Ser-402 and nearby hydrophobic amino acids (residues 402-407). Trp-452 also binds to the furanose ring of FBP, which allows for a stronger interaction between the pyruvate kinase and FBP (1).
Without the presence of FBP, pyruvate kinase forms a slightly different conformation (PDB ID: 1A3X). Residues 446-452, which surround the active site in the C domain, become more disordered in the absence of FBP and become ordered when FBP binds (1,4). The conformation without FBP is functionally very similar to pyruvate kinase complexed with FBP, with the only differences in rate of reaction and kinetics, based on the specific conditions and isozyme. However, in mammals, this difference is more apparent with the four possible isozymes, since only 2 are allosterically regulated by FBP and the other two are not (1, 4, 6).
The Dali database compares the tertiary structures of proteins by comparing folding through measuring intermolecular distances and calculating a Z-score with a sum-of-pairs method. This databse was used to find similar proteins to pyruvate kinase, with higher Z-scores indicating greater similarity (7). The PSI-BLAST database, which compares proteins with similar sequences was also used to find a similar protein based on gaps in homology between proteins, with lower E values indicating more similarity through homology (8). 4-hydroxy-2-oxo-heptane-1,7-dioate aldolase (PDB ID: 4B5X) from Escherichia coli has an E value of 2e-5, which is lower than the statistically significant criteria of being lower than 0.05, and a Z score of 18.8, which is higher than the statistically significant threshold of being above 2, in comparison to pyruvate kinase indicating high similarity in structure (7,8).
4-hydroxy-2-oxo-heptane-1,7-dioate aldolase (Hpal) is a metal dependent class II pyruvate aldolase and acts as a lyase. Hpal catalyzes the last step in 4-hydroxy-phenylacetate catabolsim in E. coli. Hpal helps transform the substrate 4-hydroxy-2-ketoheptane-1,7-dioate (HKHD) into pyruvate and succinic semialdehyde through a retro-aldol reaction. Hpa1 only has 524 residues, while pyruvate kinase has 1,000 residues. The secondary structures are similar by percent composition: Hpal is 42% helical (13 helices, 111 residues) and 15% β-sheets (8 strands, 41 residues), and pyruvate kinase was 38% helical and 18% β sheets, with double the numbers of actual secondary structures, which matches proportionally with the overall sequence lengths. Hpal and pyruvate kinase have similar electrostatic pockets and hydrogen boding. They both also have 2 identical subunits (A and B), and form dimers and tetramers (1,4,9,10).
Structurally, the active site location and ligands are different. In Hpal, Ala-174, Glu-175, Glu-147, and Arg-70 form the active site. While the positions are different, glutamine, arginine and asparagine are key amino acids in forming active sites of both pyruvate kinase and Hpal. Glutamine and asparagine also create the binding site for the bivalent metal ion. In Hpal, the ligands are a phosphate ion (PO4-) and glycerol (GOL), with a cobalt (II) ion as a cofactor. However, in pyruvate kinase the metal ion, Mn2+, is a prosthetic group that is more permanently bound and helps activates the enzyme. In Hpal, the metal ion Co2+is a cofactor that dissociates easily (1,2,9,11). Despite the different roles functionally, both pyruvate kinase and Hpal involve pyruvate and their secondary and tertiary structures reflect this similarity, especially for the domains containing the active sites to allow for substrate specificity (1,7,9).