Phosphoglycerate Kinase
Created by Vietvuong Vo
Phosphoglycerate kinase (PGK) is one of two enzymes required for ATP generation in the Calvin cycle. It is used in the seventh step of glycolysis and is classified as a transferase (1). As such, it transfers a phosphoryl group from the acyl phosphate of 1,3-diphosphoglycerate (1,3-DPG) to ADP and forms ATP and 3-phosphoglycerate (3-PGA). This reaction is essential in most cells for the production of ATP in aerobic respirators, for fermentation in anaerobic respirators, and for the fixation of carbon in photosynthetic organisms (1). PGK’s sequence has been highly conserved throughout evolution, and it can be found in all life forms, because these proteins with similar function were necessary for survival. Phosphoglycerate kinase deficiency is associated with hemolytic anemia, which is a disorder where the number of red blood cells are reduced due to hemolysis, the abnormal breakdown of red blood cells. PGK deficiency is also associated with mental retardation and epilepsy in humans (1). Isozymes, which are enzymes that differ in the primary structure but catalyze the same chemical reaction, include phosphoglycerate kinase 1 and phosphoglycerate kinase 2. The specific protein of interest has a PDB ID of 3PGK, and originates from Saccharomyces cerevisiae. Limited research has been done on 3PGK to produce a comprehensive understanding of this protein’s function and mechanism deduced from its structure, but there is a plethora of information on related proteins and their structure. Conserved domains may result in conserved functions. By understanding the structural make-up of 3PGK, it may be possible to deduce its function and pathway of catalysis, and gain a deeper understanding of biochemical processes.
There are many resources to obtain sequences. One of them is PDB, which also gives primary sources, enzyme classifications, length of AA sequences, associated ligands, modified residues, 3D representations, and more. The structure of yeast phosphoglycerate kinase has been determined with data obtained from amino acid sequence, nucleotide sequence, and X-ray crystallographic studies (X-ray diffraction at 2.5 nm resolution) (1). The substrate binding sites, as deduced from electron density maps, are compatible with known substrate specificity and the stereochemical requirements for the enzymatic reaction. 3PGK is classified as an alpha and beta protein and is part of the phosphoglycerate kinase superfamily. It is a two-domain protein and each domain is composed of six repeats of an α/β structural motif. This is a signature pattern for PGK's.(2)
Many resources have quantified the physical nature of many proteins. ProtParam, for example, provides physical and chemical parameters such as size, secondary structure, estimated half life, molecular weight and theoretical PI. 3PGK is a monomeric enzyme composed of 416 amino acids. It suggests that phosphoglycerate kinase activity is observed from residues 2-416, ATP binding from 371-374, substrate binding from 24-26 and also from 63-66. The estimated molecular weight was determined to be 44607.2 Da and the theoretical pI was determined to be 7.09 (2). This is different from the Expasy calculated result of a molecular weight of 44675.54 Da and an isoelectric point at 7.25 (3). Uniprot, another database, gives a list of general information on structure and function of proteins. The protein has an ascension number of P00560 and is present with 314,000 molecules/cell in log phase SD medium (4). ProtParam summed up the number of charged molecules. The total number of negatively charged residues (Asp + Glu) was 55. The total number of positively charged residues (Arg + Lys) was also 55. Thus, it makes the more neutral pI logical. The protein’s aliphatic index is 97.06, which is a measure of the relative volume occupied by aliphatic side chains. The protein consists of one subunit, but at the core of each domain is a 6-stranded parallel beta-sheet surrounded by alpha helices. The subunit transfers a phosphoryl group from the acly phosphate of 1,3-diphosphoglycerate (1,3-DPG) to ADP and forms ATP and 3-phosphoglycerate (3-PGA). Phosphoglycerate kinase is 34% helical, 11% beta sheets, and 55% random coils. Residues that are involved with hydrogen bonds include Asn-6 Leu-311 Gly-338 Glu-341. These bonds create tight binding of the substrate and stabilize the transition state.(2)
Important residues have been identified by the primary PDB literature source written by Watson et al. Gyl-211 is important as it works with aliphatic side chains to define the boundaries of the adenine binding hollow. Asp-372 has a carboxyl group which binds a ribose and links it to a Mg ion, which is necessary for domain movement. The residues from 210 to 212 (212 is alanine), 234 to 236, 369 to 371, 392 to 394 are consecutive glycine residues. These side chain-free regions facilitate conformational changes that are required to fulfill the protein's catalytic function. Histidines 62 and170 and arginine 21 is very similar to that found at the active site of yeast phosphoglycerate mutase. The adenine nitrogen (N6) forms a hydrogen bond with the main chain carbonyl group of residue 311 and sits on top of a conserved glycine residue 338. The triose substrate is maximally bonded to the protein and in van der Waals contact with the three glycine residues from residues 392 to 394. The imidazole group of histidine 388, extends towards the carboxyl group of glutamate 190 and forms part of the amino terminal domain. (4)
Sites like NCBI and ISI Web of knowledge are great resources to find primary research articles about a protein. Most papers focus on a single structural or functional aspect, but when many articles are viewed as a whole, a greater understanding of the big picture will result. In his paper, Blake finds that the most interesting aspect about phosphoglycerate kinase is that it consists of a single polypeptide chain, which is organized into two separated domains composed of the N-terminal and C-terminal halves of the chain. Studies of substrate binding suggest that the nucleotide and phosphoglycerate substrates are bound to the C-domain and N-domain, respectively. It was interesting to see that these sites were separated by about 12 angstroms. In order to bring the two substrates together for catalysis, a hinge-bending conformational change involving helix rotation has been proposed (5).
In another study, Wilson shows that there is a mobile section of phosphoglycerate kinase associated with the inter-domain region of the molecule. This region gives relatively well resolved, distinct resonances relative to the remainder of the protein. This suggests that the molecule fluctuates between many conformations. He predicts PGK to have several open or substrate binding forms in addition to the closed and catalytic form of the enzyme. The state the protein takes appears to be affected by the substrates and their resulting products and also several anions including sulphate, phosphate and cobalticyanide (6). More specifically, as described in X-ray studies, the pyridoxyl group of AdoP2Pxy cannot reach Lys-385 for Schiff-base formation. Labeled Lys-385 is on a beta-turn immediately following helix XII, which was suggested to interact with the nucleotide and become ordered at the active site of 3-phosphoglycerate kinase (7). Using small angle X-ray scattering from solutions of yeast phosphoglycerate kinase, Engelman and Steitz have found that the radius of gyration of the enzyme first in the presence and then in the absence of ligands, MgATP and 3-phosphoglycerate, decreases by 1.09 +/- 0.34 A. For the separate binding of MgATP, smaller decreases were observed (0.30 +/- 0.50 A). It has been computationally estimated that a substrate-induced cleft closure in PGK resulting from one lobe rotating 8-14 degrees relative to the other lobe is consistent with this observed change in radius of gyration. They suggest that the conformational change that results in the smaller radius of gyration for the ternary complex is a hinge motion of the two lobes, which closes the cleft between the two lobes. The ligand-induced change in PGK is similar to the cleft closure in hexokinase. This makes one curious to see if this special conformational change can be generalized to other kinases. (8)
Uniprot has the binding sites of substrates, products, metal ions on file. There is nucleotide binding at Gly-371, Gly-372, Asp-373, and Thr-374. There is substrate binding at Asp-24, Phe-25, and Asn-26. There is another substrate binding site at His-63, Lys-64, Gly-65, and Arg-66. (2)
Ligands are substances that form complexes with proteins. 3-phosphoglyceric acid (3PG) is a metabolic intermediate in both glycolysis and the Calvin cycle. PGK transfers a phosphoryl group from the acyl phosphate of 1,3-diphosphoglycerate, producing ATP and 3PG. ATP, Mg+2, and 3PG were all bound in crystal structure displayed on the protein data bank. Adenosine-5'-triphosphate (ATP) is significant as its stored free energy can be harnessed by a living system to do work. ATP is a ribonucleotide adenosine that carries three phosphate groups esterified to a sugar moiety. It is the cell's source for energy and phosphate. ATP is required for domain movement in 3PGK. Magnesium ions (Mg+2) are biologically ubiquitous. ATP has several negatively charged groups in neutral solution, it can chelate metals with very high affinity. ATP exists in the cell mostly in a complex with Mg2+ which changes the Gibbs free energy for reaction. Like ATP, it is required for domain movement. When the ligands bind to the protein, conformation changes are common. The structure of 3PGK is the open form of the enzyme, before substrates bind. The closed form of phosphoglycerate kinase has the PDB ID 1VPE (2).
BLAST helps one find specific proteins that are similar in sequence to a protein of interest by comparing how closely the protein’s AA sequence aligns with a comparison protein's AA sequence. These comparisons can be used to locate conserved domains. It is necessary to look at conserved residues to understand the function of a protein. PGK has the following domains with an E-value max of 0.01: [cd00318], [PTZ00005], [pfam00162], [PRK00073], [COG0126], [Carbohydrate transport and metabolism], [PLN02282], [PRK13962], and [PLN03034] (9). These conserved domains include: substrate binding site, which is well described by Sherman et al. in their research. The substrate domain consists of a cluster of conserved histidines and arginines (His-62, His-167, Arg-21, Arg-38, and Arg-168) in PGK. It has been implicated as involved in the binding of 3-phosphoglycerate (3PG) and/or stabilization of the negatively charged transition state. Site-directed mutagenesis was employed to determine if these residues were directly involved in the catalytic function of yeast PGK. Substitution of the corresponding AA by R38A and R38Q mutations resulted in a complete loss of catalytic activity. These results show that only Arg-38, of all the basic residues studied, is essential for the catalytic function of PGK. A moderate decrease in the catalytic efficiency as the result of the R21A, H167S, and R168Q mutations and an increased catalytic efficiency of the H62Q mutant rule out a possible role of a positive charge at these positions in the mechanism of phosphoryl transfer reaction. The results suggest that PGK has two binding sites for anionic ligands. These are the catalytic and regulatory sites for each substrate and the activatory and inhibitory sites for sulfate, which acts as a regulator. This implies that Arg-21, Arg-168, and His-167 are located in the activatory anion binding site. The increased Km values for both substrates and decreased specific activities of the mutants suggest that the regulatory site is close to the catalytic site (10). The idea that the ADP-regulatory and catalytic sites are next to each other is supported by Szilágyi et al. They suggest that PGK is a typical kinase with two structural domains. The domains each bind one of the two substrates, 3PG and MgATP. For the phospho-transfer reaction to take place, the substrates must be brought closer by a hinge-bending domain which was supported by Engelman and Steitz . Open and closed structures of the enzyme with different relative domain positions have been determined from different species, but a comprehensive description of this conformational transition is yet to be attained. Crystals of pig muscle PGK (1VJD) in complex with MgADP and 3PG were grown under the same conditions that produced catalytically competent Trypanosoma brucei PGK (16PK). The X-ray structure of the pig muscle ternary complex was determined at 1.8 A. Contrary to expectation, however, it represents an open conformation compared to that of T. brucei PGK. In addition, the beta-phosphate group of ADP is mobile in the new structure, in contrast to its well-defined position in T. brucei PGK. An extensive comparison was conducted to establish general differences between the two conformations. A second pair of the open and closed structures was also compared. These results propose several novel characteristic changes which follow the structural transition: “(1) the operation of a hinge at beta-strand L in the inter-domain region which greatly affects the relative domain positions; (2) the rearrangement and movement of helix 8, regulated through the interactions with the nucleotide phosphate; and (3) the existence of another hinge between helix 14 and the rest of the C-terminal part of the chain, which allows fine adjustment of the N-domain position. The main hinge at beta-strand L acts in concert with the C-terminal hinge at helix 7 described previously. Simultaneous interactions of the nucleotide phosphate groups with the loop that precedes helix 8, beta-strand J and the N terminus of helix 13 are required for propagation of the nucleotide effect towards the beta-strand L molecular hinge.” (10) This pathway describes mechanism of catalysis. Knowing this mechanism can help scientists gain valuable information about how the catalytic process occurs, and may be able to manipulate its activity to reduce the harmful effects caused by the lack of the protein.
Comparing two similar proteins may give insight to conserved structures and even function. BLAST was one tool, and it compared sequences whereas DALI compares a protein’s 3D structure with another protein’s 3D structure. A low E score suggests the primary sequences are a close match. Likewise, a low Z score, indicates that there is little difference between the proteins’ 3D conformation. Phosphoglycerate kinase from Plasmodium falcaparum (1LTK) had a E value of 1x10-145 and a Z Score: 40.2 (9, 11). It is surprising to see such similar sequence, yet a significantly different 3D structure. The ligands of PGK from P. falcaparum are: adenosine monophosphate, glycerol, and sulfate ion, which is much different than the substrates of yeast PGK. The difference in 3D structure probably means that even though the two proteins have similar sequences, their functions or reaction mechanisms may be totally different. Phosphoglycerate kinase from P. falcaparum contains 3 chains, whereas the one from yeast only has 1 chain. The amino acid length of yeast PGK is 416, but in P. Falcaparum, the aa sequence is 425 residues. Since the Z value between the two was 40.2, this means that they fold differently in space. Phosphoglycerate kinase from Thermus caldophilus (16PK) has a Z score of 37.4 and an E value: 4x10-77 (9, 11). Associated ligands are 3-phosphoglycerate, ATP, and Mg+2. This second comparison protein probably functioned more like 3PGK because the ligands are the same, which means having a similar substrate binding site and similar mechanism of reaction. Using Metacyc, we can compare the metabolic pathways and kinetics of related proteins as they may be similar to the protein under scrutiny. The enzymatic reaction of Spinacia oleracea PGK is the same as any other PGK: 3-phospho-D-glycerate + ATP <=> 1,3-bisphospho-D-glycerate + ADP. This reaction is reversible. It is observed in the following pathways: superpathway of cytosolic glycolysis in plants, pyruvate dehydrogenase and TCA cycle, sucrose degradation to ethanol and lactate for anaerobic respiration, glycolysis IV in plant cytosol, and sucrose biosynthesis I. The information from Metacyc says the protein does not follow Michaelis-Menten kinetics for this reaction. The kinetics are biphasic and the enzyme exhibits 4 to 6 times higher affinity for the substrate in the lower concentration range than in the higher range. At the low concentrations range, Km of MgATP was 240 μM and Km of 3PGA was 280 μM. At the high concentration range, Km of MgATP was 1460 μM and Km of 3PGA was 1220 μM. Inhibitors include ATP at high concentrations, so ATP would be the primary physiological regulator of enzyme activity. Km for 3-phospho-D-glycerate was 280 μM and Km for ATP was 240 μM. The optimal pH was found to be 7.5. (12) These kinetic parameters may be generalized across species. The same protein from yeast may follow these same kinetic parameters if they are conserved enough.
The enzyme exists as a monomer containing two nearly equal-sized domains that correspond to the N- and C-termini of the protein. 3-phosphoglycerate (3PG) binds to the N-terminal, while the nucleotide substrates, MgATP or MgADP, bind to the C-terminal domain of the enzyme. This extended two-domain structure is associated with large-scale 'hinge-bending' conformational changes. By comparing 3PGK wtih proteins similar in structure or sequence, the catalytic mechanism of the protein of interest was deduced. The hinge-bending domians are conserved throughout PGKs and are important for catalysis. Catalysis is not possible without the domain movements made possible by the conserved residues aforementioned. Assays can be conducted to test these hypotheses.