scholarly journals Mutational Analysis of the Binding of Alternative Substrates and Inhibitors to the Active Site of Human Glutathione Transferase P1–1

Processes ◽  
2020 ◽  
Vol 8 (10) ◽  
pp. 1232
Author(s):  
Abeer Shokeer ◽  
Aram Ismail ◽  
Usama M. Hegazy ◽  
Rüdiger H. Kolm ◽  
Bengt Mannervik
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Glutathione Transferase ◽  
Mutational Analysis ◽  
Critical Role ◽  
Catalytic Efficiency ◽  
Binding Energies ◽  
Nucleophilic Attack ◽  
Therapeutic Drugs ◽  
Glutathione Binding

Glutathione transferases (GSTs) are enzymes that play a critical role in cellular detoxication by catalyzing the nucleophilic attack of glutathione on the electrophilic center of a number of xenobiotic compounds, including many therapeutic drugs. Mutations of amino acid residues in the glutathione-binding site of human glutathione transferase P1–1, namely W39C, K45A, Q52A, Q52K, and Q52E, have been engineered. The recombinant mutant proteins were expressed in Escherichia coli, but only mutants K45A, Q52A, and Q52K showed measurable activity. Steady-state kinetics comparing glutathione with the alternative thiol substrate γ-glutamylcysteine demonstrated the importance of the glycine residue in glutathione for high catalytic efficiency. Inhibition experiments with a set of glutathione analogs structurally related to the therapeutic drugs Telintra and Telcyta enabled determination of binding energies that were contributed by different substituents. The effects of substituting amino acid side chains in the glutathione-binding site of the enzyme on binding the glutathione derivatives and catalysis were evaluated.

scholarly journals Human glutathione transferase A4-4: an Alpha class enzyme with high catalytic efficiency in the conjugation of 4-hydroxynonenal and other genotoxic products of lipid peroxidation

1998 ◽  
Vol 330 (1) ◽  
pp. 175-179 ◽  
Author(s):  
Ina HUBATSCH ◽  
Marianne RIDDERSTRÖM ◽  
Bengt MANNERVIK
Keyword(s):  
Lipid Peroxidation ◽  
Amino Acid ◽  
Glutathione Transferase ◽  
Fetal Brain ◽  
Catalytic Efficiency ◽  
Protective Role ◽  
Significant Feature ◽  
Brain Cdna Library ◽  
A Subunit ◽  
High Level

A sequence encoding a novel glutathione transferase, GST A4-4, has been identified in a human fetal brain cDNA library. The protein has been produced in Escherichia coli after optimization of the codon usage for high-level heterologous expression. The dimeric protein has a subunit molecular mass of 25704 Da based on the deduced amino acid composition. Human GST A4-4 is a member of the Alpha class but shows only 53% amino acid sequence identity with the major liver enzyme GST A1-1. High catalytic efficiency with 4-hydroxyalkenals and other cytotoxic and mutagenic products of radical reactions and lipid peroxidation is a significant feature of GST A4-4. The kcat/Km values for 4-hydroxynonenal and 4-hydroxydecenal are > 3×106 M-1·s-1, several orders of magnitude higher than the values for conventional GST substrates. 4-Hydroxynonenal and other reactive electrophiles produced by oxidative metabolism have been linked to aging, atherosclerosis, cataract formation, Parkinson's disease and Alzheimer's disease, as well as other degenerative human conditions, suggesting that human GST A4-4 fulfills an important protective role and that variations in its expression may have significant pathophysiological consequences.

scholarly journals Mutational analysis and an alternatively spliced product of B7 defines its CD28/CTLA4-binding site on immunoglobulin C-like domain.

1995 ◽  
Vol 181 (4) ◽  
pp. 1345-1355 ◽  
Author(s):  
Y Guo ◽  
Y Wu ◽  
M Zhao ◽  
X P Kong ◽  
Y Liu
Keyword(s):  
Amino Acids ◽  
T Cell ◽  
Binding Site ◽  
Mutational Analysis ◽  
Critical Role ◽  
Surface Receptors ◽  
Naturally Occurring ◽  
Alternatively Spliced ◽  
Single Binding Site

Costimulatory molecules B7 and B7-2 interact with T cell surface receptors CD28/CTLA4 and deliver a costimulatory signal essential for T cell growth. However, the structure basis of this interaction is not known. B7 and B7-2 are members of immunoglobulin (Ig) superfamily and their extracellular portion consists of an IgV- and IgC-like domain. Here we report that a naturally occurring, alternatively spliced form of B7 reveals that exon 3-encoded IgC domain is essential for CD28/CTLA4 binding. Mutational analysis of B7 demonstrates a critical role of several amino acids around loops between strands B and C and D and E, for binding CTLA4/CD28. These amino acids are clustered to form a single binding site centered at 201Y. A comparison of the effects of mutations on the binding of CD28 and CTLA4 reveals that CD28 and CTLA4 binds to the same site on B7. These results have important implications on the role of CTLA4 and CD28 in T cell costimulation. The structure of the CD28/CTLA4-binding site also provides valuable information for immune intervention targeted at the B7/B7-2-CD28/CTLA4 interactions.

scholarly journals Key amino acid residues of neuraminidase involved in influenza A virus entry

2019 ◽  
Vol 77 (6) ◽  
Author(s):  
Fangzhao Chen ◽  
Teng Liu ◽  
Jiagui Xu ◽  
Yingna Huang ◽  
Shuwen Liu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Influenza A Virus ◽  
Viral Entry ◽  
Influenza A ◽  
Mutational Analysis ◽  
Critical Role ◽  
Target Cells ◽  
Amino Acid Residues ◽  
Influenza Virus Neuraminidase

ABSTRACT Generally, influenza virus neuraminidase (NA) plays a critical role in the release stage of influenza virus. Recently, it has been found that NA may promote influenza virus to access the target cells. However, the mechanism remain unclear. Here, we reported that peramivir indeed possessed anti-influenza A virus (IAV) activity in the stage of viral entry. Importantly, we verified the critical residues of influenza NA involved in the viral entry. As a result, peramivir as an efficient NA inhibitor could suppress the initiation of IAV infection. Furthermore, mutational analysis showed NA might be associated with viral entry via amino acids residues R118, E119, D151, R152, W178, I222, E227, E276, R292 and R371. Our results demonstrated NA must contain the key amino acid residues can involve in IAV entry.

scholarly journals Proposed Mechanism for Monomethylarsonate Reductase Activity of Human Omega-class Glutathione Transferase GSTO1

2021 ◽  
Author(s):  
Aaron J Oakley
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Binding Sites ◽  
Glutathione Transferase ◽  
Reductase Activity ◽  
Inorganic Arsenic ◽  
Glutathione Transferases ◽  
Public Health Issue ◽  
Major Public Health Issue ◽  
Glutathione Binding

Contamination of drinking water with toxic inorganic arsenic is a major public health issue. The mechanisms of enzymes and transporters in arsenic elimination are therefore of interest. The human omega-class glutathione transferases have been previously shown to possess monomethylarsonate (V) reductase activity. To further understanding of this activity, molecular dynamics of human GSTO1-1 bound to glutathione with a monomethylarsonate isostere were simulated to reveal putative monomethylarsonate binding sites on the enzyme. The major binding site is in the active site, adjacent to the glutathione binding site. Based on this and previously reported biochemical data, a reaction mechanism for this enzyme is proposed. Further insights were gained from comparison of the human omega-class GSTs to homologs from a range of animals.

Adaptor protein Lnk associates with Tyr568 in c-Kit

2008 ◽  
Vol 415 (2) ◽  
pp. 241-245 ◽  
Author(s):  
Saskia Gueller ◽  
Sigal Gery ◽  
Verena Nowak ◽  
Liqin Liu ◽  
Hubert Serve ◽  
...  
Keyword(s):  
Binding Site ◽  
Glutathione Transferase ◽  
Critical Role ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Erythropoietin Receptor ◽  
Cytokine Receptors ◽  
Juxtamembrane Domain ◽  
Model Studies ◽  
Haemopoietic Cells

The adaptor protein Lnk is expressed in haemopoietic cells and plays a critical role in haemopoiesis. Animal model studies demonstrated that Lnk acts as a broad inhibitor of signalling pathways in haemopoietic lineages. Lnk belongs to a family of proteins sharing several structural motifs, including an SH2 (Src homology 2) domain which binds phosphotyrosine residues in various signal-transducing proteins. The SH2 domain is essential for Lnk-mediated negative regulation of several cytokine receptors [e.g. Mpl, EpoR (erythropoietin receptor), c-Kit]. Therefore inhibition of the binding of Lnk to cytokine receptors might lead to enhanced downstream signalling of the receptor and thereby to improved haemopoiesis in response to exposure to cytokines (e.g. erythropoietin in anaemic patients). This hypothesis led us to define the exact binding site of Lnk to the stem cell factor receptor c-Kit. Pull-down experiments using GST (glutathione transferase)-fusion proteins of the different domains of c-Kit showed that Lnk almost exclusively binds to the phosphorylated juxtamembrane domain. Binding of Lnk to the juxtamembrane domain was abolished by point mutation of Tyr568 and was competed by peptides with a phosphotyrosine residue at position 568. Co-immunoprecipitation with full-length wild-type or Y568F mutant c-Kit and Lnk confirmed these results, thus showing the importance of this phosphorylated tyrosine residue. Lnk bound directly to c-Kit without requiring other interacting partners. The identification of the binding site of Lnk to c-Kit will be useful to discover inhibitory molecules that prevent the binding of these two proteins, thus making haemopoietic cells more sensitive to growth factors.

scholarly journals Mutational analysis of Lys65 of HIV-1 reverse transcriptase

2000 ◽  
Vol 348 (1) ◽  
pp. 77-82 ◽  
Author(s):  
Nicolas SLUIS-CREMER ◽  
Dominique ARION ◽  
Neerja KAUSHIK ◽  
Henry LIM ◽  
Michael A. PARNIAK
Keyword(s):  
Amino Acid ◽  
Reverse Transcriptase ◽  
Mutational Analysis ◽  
Catalytic Efficiency ◽  
Salt Bridge ◽  
Polymerase Activity ◽  
Ph Optimum ◽  
Modelling Studies ◽  
Dntp Binding ◽  
Hiv 1

Amino acid Lys65 is part of the highly flexible β3-β4 loop in the fingers domain of the 66 kDa subunit of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Recent crystal data show that the ϵ-amino group of Lys65 interacts with the γ-phosphate of the bound deoxynucleoside triphosphate (‘dNTP’) substrate [Huang, Chopra, Verdine and Harrison (1998) Science 282, 1669-1675]. In order to biochemically define the function of RT Lys65, we have used site-specific mutagenesis to generate RT with a variety of substitutions at this position, including K65E, K65Q, K65A and K65R. Kinetic analyses demonstrate that if Lys65 in RT is substituted with an amino acid other than arginine the enzyme exhibits dramatic decreases in the binding affinity (Km) for all dNTP substrates, in RT catalytic efficiency (kcat/Km) and in the mutant enzyme's ability to carry out pyrophosphorolysis, the reverse reaction of DNA synthesis. The pH optimum for the DNA polymerase activity of K65E RT was 6.5, compared to 7.5 for the wild-type enzyme, and 8.0 for the K65R, K65A and K65Q mutants. Molecular modelling studies show that mutations of Lys65 do not affect the geometry of the loop's α-carbon backbone, but rather lead to changes in positioning of the side chains of residues Lys70 and Arg72. In particular, Glu in K65E can form a salt bridge with Arg72, leading to the diminution of the latter residue's interaction with the α-phosphate of the dNTP residue. This alteration in dNTP-binding may explain the large pH-dependent changes in both dNTP-binding and catalytic efficiency noted with the enzyme. Furthermore, the K65A, K65Q and K65E mutant enzymes are 100-fold less sensitive to all dideoxynucleoside triphosphate (‘ddNTP’) inhibitors, whereas the K65R mutation results in a selective 10-fold decrease in binding of ddCTP and ddATP only. This implies that mutations at position 65 in HIV-1 RT influence the nucleotide-binding specificity of the enzyme.

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