Mutational Analysis of the Binding of Alternative Substrates and Inhibitors to the Active Site of Human Glutathione Transferase Pl−1

Mutational Analysis of the Binding of Alternative Substrates and Inhibitors to the Active Site of Human Glutathione Transferase Pl−1

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-b...

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Main Authors: Abeer Shokeer, Aram Ismail, Usama M. Hegazy, Rüdiger H. Kolm, Bengt Mannervik
Format: Article
Language:English
Published: MDPI AG 2020-10-01
Series:Processes
Subjects:
Glutathione transferase P1–1
Telintra
Telcyta
alternative thiol substrates
inhibitor binding energy
Online Access:https://www.mdpi.com/2227-9717/8/10/1232
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spelling doaj-49fb9c8793e14fb19ab06b0af504c3652020-11-25T04:02:42ZengMDPI AGProcesses2227-97172020-10-0181232123210.3390/pr8101232Mutational Analysis of the Binding of Alternative Substrates and Inhibitors to the Active Site of Human Glutathione Transferase Pl−1Abeer Shokeer0Aram Ismail1Usama M. Hegazy2Rüdiger H. Kolm3Bengt Mannervik4Department of Molecular Biology, Genetic Engineering and Biotechnology Research Division, National Research Centre, Dokki, Giza 126 22, EgyptDepartment of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, SwedenDepartment of Molecular Biology, Genetic Engineering and Biotechnology Research Division, National Research Centre, Dokki, Giza 126 22, EgyptDepartment of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, SwedenDepartment of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, SwedenGlutathione 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 Pl−1, namely W39C, K45A, Q52A, Q52K, and Q52E, have been engineered. The recombinant mutant proteins were expressed in<i> Escherichia coli</i>, 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.https://www.mdpi.com/2227-9717/8/10/1232Glutathione transferase P1–1TelintraTelcytaalternative thiol substratesinhibitor binding energy
collection DOAJ
language English
format Article
sources DOAJ
author Abeer Shokeer
Aram Ismail
Usama M. Hegazy
Rüdiger H. Kolm
Bengt Mannervik
spellingShingle Abeer Shokeer
Aram Ismail
Usama M. Hegazy
Rüdiger H. Kolm
Bengt Mannervik
Mutational Analysis of the Binding of Alternative Substrates and Inhibitors to the Active Site of Human Glutathione Transferase Pl−1
Processes
Glutathione transferase P1–1
Telintra
Telcyta
alternative thiol substrates
inhibitor binding energy
author_facet Abeer Shokeer
Aram Ismail
Usama M. Hegazy
Rüdiger H. Kolm
Bengt Mannervik
author_sort Abeer Shokeer
title Mutational Analysis of the Binding of Alternative Substrates and Inhibitors to the Active Site of Human Glutathione Transferase Pl−1
title_short Mutational Analysis of the Binding of Alternative Substrates and Inhibitors to the Active Site of Human Glutathione Transferase Pl−1
title_full Mutational Analysis of the Binding of Alternative Substrates and Inhibitors to the Active Site of Human Glutathione Transferase Pl−1
title_fullStr Mutational Analysis of the Binding of Alternative Substrates and Inhibitors to the Active Site of Human Glutathione Transferase Pl−1
title_full_unstemmed Mutational Analysis of the Binding of Alternative Substrates and Inhibitors to the Active Site of Human Glutathione Transferase Pl−1
title_sort mutational analysis of the binding of alternative substrates and inhibitors to the active site of human glutathione transferase pl−1
publisher MDPI AG
series Processes
issn 2227-9717
publishDate 2020-10-01
description 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 Pl−1, namely W39C, K45A, Q52A, Q52K, and Q52E, have been engineered. The recombinant mutant proteins were expressed in<i> Escherichia coli</i>, 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.
topic Glutathione transferase P1–1
Telintra
Telcyta
alternative thiol substrates
inhibitor binding energy
url https://www.mdpi.com/2227-9717/8/10/1232
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AT rudigerhkolm mutationalanalysisofthebindingofalternativesubstratesandinhibitorstotheactivesiteofhumanglutathionetransferasepl1
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