Expression and Enzymatic Characterization of Human Glucose Phosphate Isomerase (GPI) Variants Accounting for GPI Deficiency

doi:10.1006/bcmd.1998.0170

Blood Cells, Molecules, and Diseases

Volume 24, Issue 1, March 1998, Pages 54-61

https://doi.org/10.1006/bcmd.1998.0170 Get rights and content

Abstract

ABSTRACT: To elucidate the structure-function relationships in glucose phosphate isomerase (GPI), we established an expression system for human GPI as a fusion protein with glutathione S-transferase (GST) in E. coli. The GST-GPI fusion protein showed affinities for the substrates glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) similar to those of the native enzyme purified from human red blood cells (RBC). We expressed GPI cDNAs with four distinct disease-causing mutations and examined their enzymatic characteristics. Although each mutation caused reduced thermal stability, an amino acid substitution Thr-5→Ile (T5I) exhibited marked thermal instability, suggesting that the amino-terminal of GPI is important for enzymatic stability. Thr-224 seemed not to be an essential residue, since the amino acid substitution Thr-224→Met (T224M) showed normal substrate affinity in spite of a slight decrease in both specific activity and thermostability. Gln-343 and Asp-539 have been shown to be in close proximity to the putative catalytic sites, and the present study showed that both Gln-343→Arg (Q343R) and Asp-539→Asn (D539N) caused impaired substrate affinity; Q343R showed high Km for both G6P and F6P, whereas D539N showed significantly decreased affinity only for F6P. These results suggest that not only reduced enzymatic stability but also impaired kinetics may disturb RBC metabolism of the GPI variants associated with hereditary hemolytic anemia.

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A novel binding of GTP stabilizes the structure and modulates the activities of human phosphoglucose isomerase/autocrine motility factor
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Citation Excerpt :
ATP is also needed for glutathione synthesis and plays a crucial role in nucleotide metabolism. Approximately thirty genetic mutations associated with this genetic disorder have been identified [16,17]; they cause either unstable proteins or proteins impaired in isomerization activity [13,18]. Without the continuous supply of PGI, due to the absence of nucleus, the red blood cells carrying the deficient versions of hPGI would have shorter lifespans, leading to haemolytic anemia.
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 K_i value of 63 μM 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.
Effects of inherited mutations on catalytic activity and structural stability of human glucose-6-phosphate isomerase expressed in Escherichia coli
2009, Biochimica et Biophysica Acta - Proteins and Proteomics
Glucose-6-phosphate isomerase (GPI), a homodimeric enzyme, catalyzes the interconversion between glucose-6-phosphate and fructose-6-phosphate. In mammals, it can also act as an autocrine motility factor, neuroleukin, and maturation factor. Deficiency of the enzymatic activity in red blood cells causes nonspherocytic hemolytic anemia in human. To gain a more complete understanding of the molecular basis for the hemolytic anemia due to the GPI-deficiency, the wild-type enzyme and sixteen genetic variants were expressed in Escherichia coli and functionally characterized. Conclusions are as follows: (1) mutations usually have negative influences on catalytic parameters, particularly k_cat, as well as structure stability; (2) mutations at or close to the active site, including R273H, H389R, and S278L, cause great damage to the catalytic function, yet those at distance can still reduce the magnitude of k_cat, despite lesser extents; (3) mutations decrease the enzyme tolerance to heat or SDS by mechanisms of decreasing packing efficiency (V101M, T195I, S278L, L487F, L339P, T375R, I525T), weakening network bonding (R75G, R347C, R347H, R472H, E495K), increasing water-accessible hydrophobic surface (R83W), and destabilizing the ternary structure (T195I, R347C, R347H, and I525T); (4) A300P, L339P, and E495K mutations may also negatively affect the protein folding efficiency.
Analysis of enzymopathies in the human red blood cells by constraint-based stoichiometric modeling approaches
2006, Computational Biology and Chemistry
The human red blood cell (RBC) metabolism is investigated by calculating steady state fluxes using constraint-based stoichiometric modeling approaches. For the normal RBC metabolism, flux balance analysis (FBA) is performed via optimization of various alternative objective functions, and the maximization of production of ATP and NADPH is found to be the primary objective of the RBC metabolism. FBA and two novel approaches, minimization of metabolic adjustment (MOMA) and regulatory on–off minimization (ROOM), which can describe the behavior of the metabolic networks in case of enzymopathies, are applied to observe the relative changes in the flux distribution of the deficient network. The deficiencies in several enzymes in RBC metabolism are investigated and the flux distributions are compared with the non-deficient FBA distribution to elucidate the metabolic changes in response to enzymopathies. It is found that the metabolism is mostly affected by the glucose-6-phosphate dehydrogenase (G6PDH) and phosphoglycerate kinase (PGK) enzymopathies, whereas the effects of the deficiency in DPGM on the metabolism are negligible. These stoichiometric modeling results are found to be in accordance with the experimental findings in the literature related to metabolic behavior of the human red blood cells, showing that human RBC metabolism can be modeled stoichiometrically.
Metabolic pathway analysis of enzyme-deficient human red blood cells
2004, BioSystems
Five enzymopathies (G6PDH, TPI, PGI, DPGM and PGK deficiencies) in the human red blood cells are investigated using a stoichiometric modeling approach, i.e., metabolic pathway analysis. Elementary flux modes (EFMs) corresponding to each enzyme deficiency case are analyzed in terms of functional capabilities. When available, experimental findings reported in literature related to metabolic behavior of the human red blood cells are compared with the results of EFM analysis. Control-effective flux (CEF) calculation, a novel approach which allows quantification and interpretation of determined EFMs, is performed for further analysis of enzymopathies. Glutathione reductase reaction is found to be the most effective reaction in terms of its CEF value in all enzymopathies in parallel with its known essential role for red blood cells. Efficiency profiles of the enzymatic reactions upon the degree of enzyme deficiency are obtained by the help of the CEF approach, as a basis for future experimental studies. CEF analysis, which is found to be promising in the analysis of erythrocyte enzymopathies, has the potential to be used in modeling efforts of human metabolism.
Functions of the conserved anionic amino acids and those interacting with the substrate phosphate group of phosphoglucose isomerase
2001, FEBS Letters
Phosphoglucose isomerase catalyzes the isomerization between glucose 6-phosphate and fructose 6-phosphate in cytoplasm, and functions as autocrine motility factor and neuroleukin outside the cells. A phosphoglucose isomerase from Bacillus stearothermophilus (pgiA) was subjected to mutagenesis study to address the catalytic function of the conserved anionic residues and those probably interacting with the phosphate group of substrates. The results suggest that Glu290 works concertedly with His311 as a general acid–base pair to initiate the isomerization step, and Glu150 assists the base function of His311. The conserved loop structure consisting of Gly205–Gly206–Arg207 plays a critical role for the recognition of substrates.
Highly heterogeneous nature of δ-aminolevulinate dehydratase (ALAD) deficiencies in ALAD porphyria
2001, Blood
Citation Excerpt :
Fusion proteins can be purified in a single step directly from bacterial lysates by using glutathione-affinity chromatography.20 The fusion proteins have also been successfully used in enzyme assays without removing GST.21–24 In this study, we purified 9 different ALAD mutants as GST-fusion proteins that had been expressed in pGEX-3X plasmid, and used them to characterize their phenotype.
The properties of 9 δ-aminolevulinate dehydratase (ALAD) mutants from patients with ALAD porphyria (ADP) were examined by bacterial expression of their complementary DNAs and by enzymologic and immunologic assays. ALADs were expressed as glutathione-S-transferase (GST) fusion proteins inEscherichia coli and purified by glutathione-affinity column chromatography. The GST-ALAD fusion proteins were recognized by anti-ALAD antibodies and were enzymatically active as ALAD. The enzymatic activities of 3 ALAD mutants, K59N, A274T, and V153M, were 69.9%, 19.3%, and 41.0% of that of the wild-type ALAD, respectively, whereas 6 mutants, G133R, K59N/G133R, F12L, R240W, V275M, and delTC, showed little activity (< 8%). These variations generally reflect the phenotype of ALAD in vivo in patients with ADP and indicate that GST-ALAD fusion protein is indeed useful for predicting of the phenotype of ALAD mutants. The location of F12L mutation in the enzyme's molecular structure indicates that its disturbance of the quaternary contact of the ALAD dimer appears to have a significant influence on the enzymatic activity. Mouse monoclonal antibodies to human ALAD were developed that specifically recognized a carboxy terminal portion of ALAD, or other regions in the enzyme. This study represents the first complete analysis of 9 mutants of ALAD identified in ADP and indicates the highly heterogeneous nature of mutations in this disorder.

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^☆: communicated by Ernest, Beutler, M.D.02/13/98

^f1: Reprint request to: Hitoshi Kanno, M.D., Ph.D., Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo 105, Japan. phone 81-3-3588-1111 Ext 4415, fax 81-3-3505-6205, email: [email protected]

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Regular ArticleExpression and Enzymatic Characterization of Human Glucose Phosphate Isomerase (GPI) Variants Accounting for GPI Deficiency☆

Abstract

Blood

Blood

Genomics

Genomics

Blood

Blood

Blood Cells Mol Dis

Blood Cells Mol Dis

Clin Chim Acta

J Biol Chem

J Biol Chem

Arch Biochem Biophys

Blood

Blood

Molecular cloning and expression of neuroleukin, a neurotrophic factor for spinal and sensory neurons

Science

Molecular basis of erythroenzymopathies associated with hereditary hemolytic anemia: Tabulation of mutant enzymes

Am J Hematol

Glucose-6-phosphate dehydrogenase (G6PD) deficiency

N Engl J Med

Regular Article
Expression and Enzymatic Characterization of Human Glucose Phosphate Isomerase (GPI) Variants Accounting for GPI Deficiency☆