Structural contributions of Delta class glutathione transferase active-site residues to catalysis

Jantana Wongsantichon, Robert C. Robinson, Albert J. Ketterman

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

GST (glutathione transferase) is a dimeric enzyme recognized for biotransformation of xenobiotics and endogenous toxic compounds. In the present study, residues forming the hydrophobic substrate-binding site (H-site) of a Delta class enzyme were investigated in detail for the first time by site-directed mutagenesis and crystallographic studies. Enzyme kinetics reveal that Tyr111 indirectly stabilizes GSH binding, Tyr119 modulates hydrophobic substrate binding and Phe123 indirectly modulates catalysis. Mutations at Tyr111 and Phe123 also showed evidence for positive co-operativity for GSH and 1-chloro-2,4- dinitrobenzene respectively, strongly suggesting a role for these residues in manipulating subunit-subunit communication. In the present paper we report crystal structures of the wild-type enzyme, and two mutants, in complex with S-hexylglutathione. This study has identified an aromatic 'zipper' in the H-site contributing a network of aromatic π-π interactions. Several residues of the cluster directly interact with the hydrophobic substrate, whereas others indirectly maintain conformational stability of the dimeric structure through the C-terminal domain (domain II). The Y119E mutant structure shows major main-chain rearrangement of domain II. This reorganization is moderated through the 'zipper' that contributes to the H-site remodelling, thus illustrating a role in co-substrate binding modulation. The F123A structure shows molecular rearrangement of the H-site in one subunit, but not the other, explaining weakened hydrophobic substrate binding and kinetic co-operativity effects of Phe123 mutations. The three crystal structures provide comprehensive evidence of the aromatic 'zipper' residues having an impact upon protein stability, catalysis and specificity.Consequently, 'zipper' residues appear to modulate and co-ordinate substrate processing through permissive flexing.

Original languageEnglish
Pages (from-to)25-32
Number of pages8
JournalBiochemical Journal
Volume428
Issue number1
DOIs
Publication statusPublished - May 15 2010
Externally publishedYes

Fingerprint

Glutathione Transferase
Catalysis
Catalytic Domain
Binding Sites
Substrates
Enzymes
Fasteners
Dinitrochlorobenzene
Mutation
Protein Stability
Poisons
Xenobiotics
Biotransformation
Site-Directed Mutagenesis
Molecular Structure
Crystal structure
Enzyme kinetics
Mutagenesis
Molecular structure
Modulation

Keywords

  • Crystal structure
  • Glutathione transferase (GST)
  • Structural motif
  • Structure-function relationship
  • Substrate specificity

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Structural contributions of Delta class glutathione transferase active-site residues to catalysis. / Wongsantichon, Jantana; Robinson, Robert C.; Ketterman, Albert J.

In: Biochemical Journal, Vol. 428, No. 1, 15.05.2010, p. 25-32.

Research output: Contribution to journalArticle

@article{04743558e2ac4b83bd723eedc2a24c5a,
title = "Structural contributions of Delta class glutathione transferase active-site residues to catalysis",
abstract = "GST (glutathione transferase) is a dimeric enzyme recognized for biotransformation of xenobiotics and endogenous toxic compounds. In the present study, residues forming the hydrophobic substrate-binding site (H-site) of a Delta class enzyme were investigated in detail for the first time by site-directed mutagenesis and crystallographic studies. Enzyme kinetics reveal that Tyr111 indirectly stabilizes GSH binding, Tyr119 modulates hydrophobic substrate binding and Phe123 indirectly modulates catalysis. Mutations at Tyr111 and Phe123 also showed evidence for positive co-operativity for GSH and 1-chloro-2,4- dinitrobenzene respectively, strongly suggesting a role for these residues in manipulating subunit-subunit communication. In the present paper we report crystal structures of the wild-type enzyme, and two mutants, in complex with S-hexylglutathione. This study has identified an aromatic 'zipper' in the H-site contributing a network of aromatic π-π interactions. Several residues of the cluster directly interact with the hydrophobic substrate, whereas others indirectly maintain conformational stability of the dimeric structure through the C-terminal domain (domain II). The Y119E mutant structure shows major main-chain rearrangement of domain II. This reorganization is moderated through the 'zipper' that contributes to the H-site remodelling, thus illustrating a role in co-substrate binding modulation. The F123A structure shows molecular rearrangement of the H-site in one subunit, but not the other, explaining weakened hydrophobic substrate binding and kinetic co-operativity effects of Phe123 mutations. The three crystal structures provide comprehensive evidence of the aromatic 'zipper' residues having an impact upon protein stability, catalysis and specificity.Consequently, 'zipper' residues appear to modulate and co-ordinate substrate processing through permissive flexing.",
keywords = "Crystal structure, Glutathione transferase (GST), Structural motif, Structure-function relationship, Substrate specificity",
author = "Jantana Wongsantichon and Robinson, {Robert C.} and Ketterman, {Albert J.}",
year = "2010",
month = "5",
day = "15",
doi = "10.1042/BJ20091939",
language = "English",
volume = "428",
pages = "25--32",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "1",

}

TY - JOUR

T1 - Structural contributions of Delta class glutathione transferase active-site residues to catalysis

AU - Wongsantichon, Jantana

AU - Robinson, Robert C.

AU - Ketterman, Albert J.

PY - 2010/5/15

Y1 - 2010/5/15

N2 - GST (glutathione transferase) is a dimeric enzyme recognized for biotransformation of xenobiotics and endogenous toxic compounds. In the present study, residues forming the hydrophobic substrate-binding site (H-site) of a Delta class enzyme were investigated in detail for the first time by site-directed mutagenesis and crystallographic studies. Enzyme kinetics reveal that Tyr111 indirectly stabilizes GSH binding, Tyr119 modulates hydrophobic substrate binding and Phe123 indirectly modulates catalysis. Mutations at Tyr111 and Phe123 also showed evidence for positive co-operativity for GSH and 1-chloro-2,4- dinitrobenzene respectively, strongly suggesting a role for these residues in manipulating subunit-subunit communication. In the present paper we report crystal structures of the wild-type enzyme, and two mutants, in complex with S-hexylglutathione. This study has identified an aromatic 'zipper' in the H-site contributing a network of aromatic π-π interactions. Several residues of the cluster directly interact with the hydrophobic substrate, whereas others indirectly maintain conformational stability of the dimeric structure through the C-terminal domain (domain II). The Y119E mutant structure shows major main-chain rearrangement of domain II. This reorganization is moderated through the 'zipper' that contributes to the H-site remodelling, thus illustrating a role in co-substrate binding modulation. The F123A structure shows molecular rearrangement of the H-site in one subunit, but not the other, explaining weakened hydrophobic substrate binding and kinetic co-operativity effects of Phe123 mutations. The three crystal structures provide comprehensive evidence of the aromatic 'zipper' residues having an impact upon protein stability, catalysis and specificity.Consequently, 'zipper' residues appear to modulate and co-ordinate substrate processing through permissive flexing.

AB - GST (glutathione transferase) is a dimeric enzyme recognized for biotransformation of xenobiotics and endogenous toxic compounds. In the present study, residues forming the hydrophobic substrate-binding site (H-site) of a Delta class enzyme were investigated in detail for the first time by site-directed mutagenesis and crystallographic studies. Enzyme kinetics reveal that Tyr111 indirectly stabilizes GSH binding, Tyr119 modulates hydrophobic substrate binding and Phe123 indirectly modulates catalysis. Mutations at Tyr111 and Phe123 also showed evidence for positive co-operativity for GSH and 1-chloro-2,4- dinitrobenzene respectively, strongly suggesting a role for these residues in manipulating subunit-subunit communication. In the present paper we report crystal structures of the wild-type enzyme, and two mutants, in complex with S-hexylglutathione. This study has identified an aromatic 'zipper' in the H-site contributing a network of aromatic π-π interactions. Several residues of the cluster directly interact with the hydrophobic substrate, whereas others indirectly maintain conformational stability of the dimeric structure through the C-terminal domain (domain II). The Y119E mutant structure shows major main-chain rearrangement of domain II. This reorganization is moderated through the 'zipper' that contributes to the H-site remodelling, thus illustrating a role in co-substrate binding modulation. The F123A structure shows molecular rearrangement of the H-site in one subunit, but not the other, explaining weakened hydrophobic substrate binding and kinetic co-operativity effects of Phe123 mutations. The three crystal structures provide comprehensive evidence of the aromatic 'zipper' residues having an impact upon protein stability, catalysis and specificity.Consequently, 'zipper' residues appear to modulate and co-ordinate substrate processing through permissive flexing.

KW - Crystal structure

KW - Glutathione transferase (GST)

KW - Structural motif

KW - Structure-function relationship

KW - Substrate specificity

UR - http://www.scopus.com/inward/record.url?scp=77953416164&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77953416164&partnerID=8YFLogxK

U2 - 10.1042/BJ20091939

DO - 10.1042/BJ20091939

M3 - Article

C2 - 20196771

AN - SCOPUS:77953416164

VL - 428

SP - 25

EP - 32

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 1

ER -