Human Phosphoglucose Isomerase Research Articles

Novel N-substituted 5-phosphate-d-arabinonamide derivatives as strong inhibitors of phosphoglucose isomerases: Synthesis, structure-activity relationship and crystallographic studies

Phosphoglucose isomerase (PGI) is a cytosolic enzyme that catalyzes the reversible interconversion of d-glucose 6-phosphate and d-fructose 6-phosphate in glycolysis. Outside the cell, PGI is also known as autocrine motility factor (AMF), a cytokine secreted by a large variety of tumor cells that stimulates motility of cancer cells in vitro and metastases development in vivo. Human PGI and AMF are strictly identical proteins both in terms of sequence and 3D structure, and AMF activity is known to involve, at least in part, the enzymatic active site. Hence, with the purpose of finding new strong AMF-PGI inhibitors that could be potentially used as anticancer agents and/or as bioreceptors for carbohydrate-based electrochemical biosensors, we report in this study the synthesis and kinetic evaluation of several new human PGI inhibitors derived from the synthon 5-phospho-d-arabinono-1,4-lactone. Although not designed as high-energy intermediate analogue inhibitors of the enzyme catalyzed isomerization reaction, several of these N-substituted 5-phosphate-d-arabinonamide derivatives appears as new strong PGI inhibitors. For one of them, we report its crystal structure in complex with human PGI at 2.38 Å. Detailed analysis of its interactions at the active site reveals a new binding mode and shows that human PGI is relatively tolerant for modified inhibitors at the “head” C-1 part, offering promising perspectives for the future design of carbohydrate-based biosensors.

Electrostatic and Chemical Properties in Natural and Designed Enzymes

Advances have been made in understanding how enzymes achieve their exquisite catalytic power. However there are still gaps in our understanding, as computationally driven enzyme design efforts are still in their infancy and must be followed by years of directed evolution in order to achieve reasonable turnover rates. The goal of this work is to understand the intrinsic properties that give natural enzymes their catalytic capabilities and to learn how to build these properties in silico, for the design and expression of artificial enzymes that catalyze reactions that do not occur in nature. Partial Order Optimum Likelihood (POOL) is a machine learning method developed by us to predict residues important for function, using the 3D structure of the query protein. The input features to POOL are based on computed electrostatic and chemical properties from THEMATICS. These input features are effectively measures of the strength of coupling between protonation events. POOL is used to characterize the properties of natural enzymes that are necessary for efficient catalysis. Catalytic sites in proteins are characterized in part by networks of strongly coupled protonation states; these networks impart the necessary electrostatic and proton‐transfer properties to the active residues in the first layer around the reacting substrate molecule(s). Typically these networks include first‐, second‐, and sometimes third‐layer residues. POOL‐predicted, multi‐layer active sites with significant participation by distal residues have been verified experimentally by single‐point site‐directed mutagenesis and kinetics assays for Ps. putida nitrile hydratase, human phosphoglucose isomerase, E. coli replicative DNA polymerase Pol III, E. coli Y family DNA polymerase DinB, ornithine transcarbamoylase, and glycinamide ribonucleotide transformylase (GART). In designed enzymes, such as retroaldolases, the residue‐specific input features to POOL – measures of the strength of coupling between protonation equilibria – rise as the enzymes evolve to higher rates of catalytic turnover. These concepts can enhance current enzyme design protocols.Support or Funding InformationSupported by NSF MCB‐1517290.

Electrostatic interactions in natural enzymes: What can we learn for enzyme design?

The goal of this work is to understand the intrinsic properties that give natural enzymes their catalytic power and to learn how to build these properties in silico, for the design and expression of artificial enzymes that catalyze reactions that do not occur in nature. Partial Order Optimum Likelihood (POOL) is a machine learning method developed by us to predict residues important for function, using the 3D structure of the query protein. The input features to POOL are based on computed electrostatic and chemical properties from THEMATICS. These input features are effectively measures of the strength of coupling between protonation events. POOL is used to characterize the properties of natural enzymes that are necessary for efficient catalysis. Catalytic sites in proteins are characterized by networks of strongly coupled protonation states; these networks impart the necessary electrostatic and proton‐transfer properties to the active residues in the first layer around the reacting substrate molecule(s). Typically these networks include first‐, second‐, and sometimes third‐layer residues. POOL‐predicted, multi‐layer active sites with significant participation by distal residues have been verified experimentally by single‐point site‐directed mutagenesis and kinetics assays for Ps. putida nitrile hydratase, human phosphoglucose isomerase, E. coli replicative DNA polymerase Pol III, E. coli Y family DNA polymerase DinB, and E. coli ornithine transcarbamoylase. In designed enzymes, such as retroaldolases, the residue‐specific input features to POOL – measures of the strength of coupling between protonation equilibria – rise as the enzymes evolve to higher rates of catalytic turnover. An approach to build these properties into the initial designs is proposed.Support or Funding InformationNSF MCB‐1517290 (MJO & PJB) and a National Institute of Justice Fellowship (TAC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel binding of GTP stabilizes the structure and modulates the activities of human phosphoglucose isomerase/autocrine motility factor

Phosphoglucose isomerase (PGI) catalyzes the interconversion between glucose 6-phosphate and fructose 6-phosphate in the glycolysis pathway. In mammals, the enzyme is also identical to the extracellular proteins neuroleukin, tumor-secreted autocrine motility factor (AMF) and differentiation and maturation mediator for myeloid leukemia. Hereditary deficiency of the enzyme causes non-spherocytic hemolytic anemia in human. In the present study, a novel interaction between GTP and human PGI was corroborated by UV-induced crosslinking, affinity purification and kinetic study. GTP not only inhibits the isomerization activity but also compromises the AMF function of the enzyme. Kinetic studies, including the Yonetani-Theorell method, suggest that GTP is a competitive inhibitor with a Ki value of 63μM and the GTP-binding site partially overlaps with the catalytic site. In addition, GTP stabilizes the structure of human PGI against heat- and detergent-induced denaturation. Molecular modelling and dynamic simulation suggest that GTP is bound in a syn-conformation with the γ-phosphate group located near the phosphate-binding loop and the ribose moiety positioned away from the active-site residues.

Prediction of distal residue participation in enzyme catalysis.

A scoring method for the prediction of catalytically important residues in enzyme structures is presented and used to examine the participation of distal residues in enzyme catalysis. Scores are based on the Partial Order Optimum Likelihood (POOL) machine learning method, using computed electrostatic properties, surface geometric features, and information obtained from the phylogenetic tree as input features. Predictions of distal residue participation in catalysis are compared with experimental kinetics data from the literature on variants of the featured enzymes; some additional kinetics measurements are reported for variants of Pseudomonas putida nitrile hydratase (ppNH) and for Escherichia coli alkaline phosphatase (AP). The multilayer active sites of P. putida nitrile hydratase and of human phosphoglucose isomerase are predicted by the POOL log ZP scores, as is the single-layer active site of P. putida ketosteroid isomerase. The log ZP score cutoff utilized here results in over-prediction of distal residue involvement in E. coli alkaline phosphatase. While fewer experimental data points are available for P. putida mandelate racemase and for human carbonic anhydrase II, the POOL log ZP scores properly predict the previously reported participation of distal residues.

A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

Structural studies of phosphoglucose isomerase fromMycobacterium tuberculosisH37Rv

Phosphoglucose isomerase (PGI) plays a key role in both glycolysis and gluconeogenesis inside the cell, whereas outside the cell it exhibits cytokine properties. PGI is also known to act as an autocrine motility factor, a neuroleukin agent and a differentiation and maturation mediator. Here, the first crystal structure of PGI from Mycobacterium tuberculosis H37Rv (Mtb) is reported. The structure was refined at 2.25 A resolution and revealed the presence of one molecule in the asymmetric unit with two globular domains. As known previously, the active site of Mtb PGI contains conserved residues including Glu356, Glu216 and His387 (where His387 is from the neighbouring molecule). The crystal structure of Mtb PGI was observed to be rather more similar to human PGI than other nonbacterial PGIs, with only a few differences being detected in the loops, arm and hook regions of the human and Mtb PGIs, suggesting that the M. tuberculosis enzyme uses the same enzyme mechanism.

Structural and functional analyses of phosphoglucose isomerase from Vibrio vulnificus and its lysyl aminopeptidase activity

Phosphoglucose isomerase (PGI) with a novel lysyl aminopeptidase (LysAP) activity was recently isolated and partially characterized from the human pathogen, Vibrio vulnificus. This PGI is a heterodimer consisting of 60.8- and 23.4-kDa subunits, which together provide LysAP activity. The present study further characterizes the complex structure and functions of Vibrio PGI and draws parallels with rabbit and human PGI. A Proscan search of Vibrio PGI revealed 194 different structural motifs of which 124 and 127 were also found in rabbit and human PGI, respectively. Vibrio PGI contains motifs for the serine, histidine and aspartic acid active sites of the subtilase family of serine proteases which form a putative catalytic triad consisting of His534 and Ser159 on the 60.8-kDa subunit and Asp53 on the 23.4-kDa subunit. Together, they form one LysAP site for each heterodimer. Each active site motif is overlapped by motifs for EF-hand calcium binding domains. The LysAP activity was inhibited by the addition of ≥10 μM Ca 2+, suggesting that the EF-hand calcium-binding domain may be a natural regulatory region for LysAP activity. In contrast, PGI's isomerase activity was enhanced at Ca 2+ concentrations >100 μM. PGI-LysAP cleaved the amino-terminal lysyl residue from des-Arg 10-kallidin producing des-Arg 9-bradykinin; therefore, Vibrio PGI-LysAP may serve as a virulence factor to enhance Vibrio invasiveness. Together, these data provide a framework to account for PGI's LysAP activity and further demonstrate the structural complexity and functional importance of this molecule.

The structure of human phosphoglucose isomerase complexed with a transition-state analogue.

Phosphoglucose isomerase (PGI) is a workhorse enzyme of carbohydrate metabolism that interconverts glucose 6-phosphate and fructose 6-phosphate. Outside the cell, however, the protein appears to function as a cytokine. A crystal structure of human PGI bound with 5-phosphoarabinonate, a strong inhibitor that mimics the cis-enediol(ate) intermediate of the reaction, has been determined at 2.5 A resolution. The structure helps to confirm the assignment of Glu357 as the base catalyst in the isomerase reaction.

The enzymatic activity of phosphoglucose isomerase is not required for its cytokine function

PGI is a housekeeping gene encoding phosphoglucose isomerase (PGI) a glycolytic enzyme that also functions as a cytokine (autocrine motility factor (AMF)/neuroleukin/maturation factor) upon secretion from the cell and binding to its 78 kDa seven-transmembrane domain receptor (gp78/AMF-R). PGI contains a CXXC motif, characteristic of redox proteins and possibly evolutionarily related to the CC and CXC motif of the chemokine gene family. Using site-directed mutagenesis, single- and double-deletion (CXC, CC) mutants were created by deleting amino acids 331 and 332 of human PGI, respectively. The mutant proteins lost their enzymatic activity; however, neither of the deletions augmented the proteins’ binding affinity to the receptor and all maintained cytokine function. The results demonstrate that the enzymatic activity of PGI is not essential for either receptor binding or cytokine function of human PGI.

Crystal structure of human phosphoglucose isomerase and analysis of the initial catalytic steps

The second enzyme in the glycolytic pathway, phosphoglucose isomerase (PGI), catalyses an intracellular aldose–ketose isomerization. Here we describe the human recombinant PGI structure (hPGI) solved in the absence of active site ligands. Crystals isomorphous to those previously reported were used to collect a 94% complete data set to a limiting resolution of 2.1 Å. From the comparison between the free active site hPGI structure and the available human and rabbit PGI (rPGI) structures, a mechanism for protein initial catalytic steps is proposed. Binding of the phosphate moiety of the substrate to two distinct elements of the active site is responsible for driving a series of structural changes resulting in the polarisation of the active site histidine, priming it for the initial ring-opening step of catalysis.

The crystal structure of human phosphoglucose isomerase at 1.6 Å resolution: implications for catalytic mechanism, cytokine activity and haemolytic anaemia

Phosphoglucose isomerase (PGI) is a multifunctional protein, which, inside the cell, functions as a housekeeping enzyme of glycolysis and gluconeogenesis and, outside the cell, exerts wholly unrelated cytokine properties. We have determined the structure of human PGI to a resolution of 1.6 Å using X-ray crystallography. The structure is highly similar to other PGIs, especially the architecture of the active site. Fortuitous binding of a sulphate molecule from the crystallisation solution has facilitated an accurate description of the substrate phosphate-binding site. Comparison with both native and inhibitor-bound rabbit PGI structures shows that two loops move closer to the active site upon binding inhibitor. Interestingly, the human structure most closely resembles the inhibitor-bound structure, suggesting that binding of the phosphate moiety of the substrate may trigger this conformational change. We suggest a new mechanism for catalysis that uses Glu357 as the base catalyst for the isomerase reaction rather than His388 as proposed previously. The human PGI structure has also provided a detailed framework with which to map mutations associated with non-spherocytic haemolytic anaemia.

Human phosphoglucose isomerase: expression, purification, crystallization and preliminary crystallographic analysis.

Phosphoglucose isomerase (PGI) is the second enzyme in the glycolytic pathway and catalyzes an aldose-ketose isomerization. Outside the cell, PGI has been found to function as both a cytokine and as a growth factor. The human pgi gene was cloned and the expressed enzyme was purified to homogeneity. Isomorphous crystals were obtained under two conditions and belong to the P2(1)2(1)2(1) space group, with unit-cell parameters a = 80.37, b = 107.54, c = 270.33 A. A 94.7% complete data set was obtained and processed to a limiting resolution of 2.6 A. The asymmetric unit contains two hPGI dimers according to density calculations, a self-rotation function map and molecular-replacement solution.

84] Phosphoglucose isomerase of human erythrocytes and cardiac tissue

This chapter describes the assay method, purification procedure, and properties of phosphoglucose isomerase of human erythroeytes and cardiac tissue. Enzyme activity can be measured in either the forward or the reverse direction by using the appropriate coupling enzymes and monitoring the oxidation or reduction of NADH or NADP spectrophotometrically. The isomerase activity is more conveniently assayed in the reverse direction by coupling with glucose-6-phosphate dehydrogenase and following the rate of reduction of NADP. The forward reaction can be measured by coupling with phosphofructokinase, aldolase, triosephosphate isomerase, and α -glycerophosphate dehydrogenase and monitoring the rate of oxidation of NADH. The purity of both substrates (fructose 6-phosphate and glucose 6-phosphate as substrates) and all coupling enzymes is adequate to prevent baseline drift or interference during the assay. In either the forward or reverse assays, the substrates are used as their sodium salts, and the concentrations of substrates are determined enzymically. The method is discussed that has been developed for the rapid isolation of human phosphoglucose isomerase by substrate elution from phosphocellulose. All steps carried out at 0–4° and 0.1% v/v 2-mercaptoethanol is included in all buffer solutions. The physical, chemical, and catalytic properties of the enzyme isolated from erythrocytes are identical with the enzyme isolated from muscle tissue. Although erythrocytes are often the only available source of material for the isolation, muscle tissue is preferred, due to its higher concentration of the enzyme.

A Point Mutation Increasing the Stability of Human Phosphoglucose Isomerase

Abstract A method has been developed for the rapid isolation of human phosphoglucose isomerase by substrate-induced elution from phosphocellulose. The high degree of selectivity of the elution provides homogeneous, crystalline enzyme from erythrocytes with a 30,000-fold purification and an overall yield of approximately 70%. A genetic variant form of the enzyme (Singh variant) has also been isolated from erythrocytes of an individual heterozygous for this rare allele. The physical, chemical, and catalytic properties of the normal wild type phosphoglucose isomerase have been compared with those of the variant protein. Sedimentation equilibrium ultracentrifugation showed that both the wild type and variant enzymes are of identical molecular weights (wild type, 132,000 ± 2,000; Singh variant, 131,000 ± 3,000) and are dimers composed of identical subunits. Although the wild type isomerase electrofocused as a single component with an isoelectric pH of 9.25, the enzyme isolated from the Singh variant erythrocytes was resolved into the wild type homodimer (pI 9.25), the mixed heterodimer (pI 9.40), and the variant homodimer (pI 9.57). The wild type and variant homodimers were S-carboxymethylated, and the lysine residues carbamylated with potassium cyanate. Following tryptic digestion, peptide maps revealed a single altered peptide from the variant isomerase. The amino acid substitution in the variant is believed to be an acidic → neutral replacement. This substitution has no effect on the catalytic properties of the enzyme. However, the variant homodimer is more stable toward heat or treatment with a variety of denaturants such as guanidine hydrochloride, urea, or sodium dodecyl sulfate than is than is the wild type enzyme. Thus, while the point mutation does not affect the binding of substrates or inhibitors, it has a readily measurable effect on the over-all stability of the protein. The increased stability of the variant may account for the increased levels of phosphoglucose isomerase activity measured in blood from individuals heterozygous for this genetic phenotype.

Qualitative and quantitative variants of human phosphoglucose isomerase.

Eight rare variants of human PGI have been discovered during a survey of 3,300 blood samples from various parts of the world. Seven of the 8 variants had altered electrophoretic mobilities. One of the

Purification and characterization of human phosphoglucose isomerase

1. 1. The glycolytic enzyme phosphoglucose isomerase ( d-glucose-6-phosphate ketol isomerase, EC 5.3.1.9) has been purified from human muscle. This preparation was homogeneous by several criteria. 2. 2. The molecular weight of the enzyme obtained by the sedimentation-equilibrium method was 134 000. Following dissociation in guanidine hydrochloride the molecular weight was 61 000, indicating a dimeric structure for the enzyme. The isoelectric point of the enzyme was determined by electrofocusing and found to be pH 8.9. 3. 3. The amino acid composition of the enzyme was determined.