Transition state model and mechanism of nucleophilic aromatic substitution reactions catalyzed by human glutathione S-transferase M1a-1a

Yury Patskovsky, Larysa Patskovska, Steven C. Almo, Irving Listowsky

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

An active site His107 residue distinguishes human glutathione S-transferase hGSTM1-1 from other mammalian Mu-class GSTs. The crystal structure of hGSTM1a-1a with bound glutathione (GSH) was solved to 1.9 Å resolution, and site-directed mutagenesis supports the conclusion that a proton transfer occurs in which bound water at the catalytic site acts as a primary proton acceptor from the GSH thiol group to transfer the proton to His107. The structure of the second substrate-binding site (H-site) was determined from hGSTM1a-1a complexed with 1-glutathionyl-2,4-dinitrobenzene (GS-DNB) formed by a reaction in the crystal between GSH and 1-chloro-2,4-dinitrobenzene (CDNB). In that structure, the GSH-binding site (G-site) is occupied by the GSH moiety of the product in the same configuration as that of the enzyme-GSH complex, and the dinitrobenzene ring is anchored between the side chains of Tyr6, Leu12, His107, Met108, and Tyr115. This orientation suggested a distinct transition state that was substantiated from the structure of hGSTM1a-1a complexed with transition state analogue 1-S-(glutathionyl)-2,4,6-trinitrocyclohexadienate (Meisenheimer complex). Kinetic data for GSTM1a-1a indicate that kcat (CDNB) for the reaction is more than 3 times greater than k cat (FDNB), even though the nonenzymatic second-order rate constant is more than 50-fold greater for 1-fluoro-2,4-dinitrobenzene (FDNB), and the product is the same for both substrates. In addition, Km (FDNB) is about 20 times less than Km (CDNB). The results are consistent with a mechanism in which the formation of the transition state is rate-limiting in the nucleophilic aromatic substitution reactions. Data obtained with active-site mutants support transition states in which Tyr115, Tyr6, and His107 side chains are involved in the stabilization of the Meisenheimer complex via interactions with the ortho nitro group of CDNB or FDNB and provide insight into the means by which GSTs adapt to accommodate different substrates.

Original languageEnglish (US)
Pages (from-to)3852-3862
Number of pages11
JournalBiochemistry
Volume45
Issue number12
DOIs
StatePublished - Mar 28 2006

Fingerprint

Glutathione Transferase
Substitution reactions
Protons
Catalytic Domain
Substrates
Binding Sites
Dinitrobenzenes
Dinitrofluorobenzene
Dinitrochlorobenzene
Mutagenesis
Proton transfer
Site-Directed Mutagenesis
2,4-dinitrobenzene
Sulfhydryl Compounds
Glutathione
Rate constants
Cats
Stabilization
Crystal structure
Crystals

ASJC Scopus subject areas

  • Biochemistry

Cite this

Transition state model and mechanism of nucleophilic aromatic substitution reactions catalyzed by human glutathione S-transferase M1a-1a. / Patskovsky, Yury; Patskovska, Larysa; Almo, Steven C.; Listowsky, Irving.

In: Biochemistry, Vol. 45, No. 12, 28.03.2006, p. 3852-3862.

Research output: Contribution to journalArticle

@article{da20a32431324d719a51860c91aed4f9,
title = "Transition state model and mechanism of nucleophilic aromatic substitution reactions catalyzed by human glutathione S-transferase M1a-1a",
abstract = "An active site His107 residue distinguishes human glutathione S-transferase hGSTM1-1 from other mammalian Mu-class GSTs. The crystal structure of hGSTM1a-1a with bound glutathione (GSH) was solved to 1.9 {\AA} resolution, and site-directed mutagenesis supports the conclusion that a proton transfer occurs in which bound water at the catalytic site acts as a primary proton acceptor from the GSH thiol group to transfer the proton to His107. The structure of the second substrate-binding site (H-site) was determined from hGSTM1a-1a complexed with 1-glutathionyl-2,4-dinitrobenzene (GS-DNB) formed by a reaction in the crystal between GSH and 1-chloro-2,4-dinitrobenzene (CDNB). In that structure, the GSH-binding site (G-site) is occupied by the GSH moiety of the product in the same configuration as that of the enzyme-GSH complex, and the dinitrobenzene ring is anchored between the side chains of Tyr6, Leu12, His107, Met108, and Tyr115. This orientation suggested a distinct transition state that was substantiated from the structure of hGSTM1a-1a complexed with transition state analogue 1-S-(glutathionyl)-2,4,6-trinitrocyclohexadienate (Meisenheimer complex). Kinetic data for GSTM1a-1a indicate that kcat (CDNB) for the reaction is more than 3 times greater than k cat (FDNB), even though the nonenzymatic second-order rate constant is more than 50-fold greater for 1-fluoro-2,4-dinitrobenzene (FDNB), and the product is the same for both substrates. In addition, Km (FDNB) is about 20 times less than Km (CDNB). The results are consistent with a mechanism in which the formation of the transition state is rate-limiting in the nucleophilic aromatic substitution reactions. Data obtained with active-site mutants support transition states in which Tyr115, Tyr6, and His107 side chains are involved in the stabilization of the Meisenheimer complex via interactions with the ortho nitro group of CDNB or FDNB and provide insight into the means by which GSTs adapt to accommodate different substrates.",
author = "Yury Patskovsky and Larysa Patskovska and Almo, {Steven C.} and Irving Listowsky",
year = "2006",
month = "3",
day = "28",
doi = "10.1021/bi051823+",
language = "English (US)",
volume = "45",
pages = "3852--3862",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "12",

}

TY - JOUR

T1 - Transition state model and mechanism of nucleophilic aromatic substitution reactions catalyzed by human glutathione S-transferase M1a-1a

AU - Patskovsky, Yury

AU - Patskovska, Larysa

AU - Almo, Steven C.

AU - Listowsky, Irving

PY - 2006/3/28

Y1 - 2006/3/28

N2 - An active site His107 residue distinguishes human glutathione S-transferase hGSTM1-1 from other mammalian Mu-class GSTs. The crystal structure of hGSTM1a-1a with bound glutathione (GSH) was solved to 1.9 Å resolution, and site-directed mutagenesis supports the conclusion that a proton transfer occurs in which bound water at the catalytic site acts as a primary proton acceptor from the GSH thiol group to transfer the proton to His107. The structure of the second substrate-binding site (H-site) was determined from hGSTM1a-1a complexed with 1-glutathionyl-2,4-dinitrobenzene (GS-DNB) formed by a reaction in the crystal between GSH and 1-chloro-2,4-dinitrobenzene (CDNB). In that structure, the GSH-binding site (G-site) is occupied by the GSH moiety of the product in the same configuration as that of the enzyme-GSH complex, and the dinitrobenzene ring is anchored between the side chains of Tyr6, Leu12, His107, Met108, and Tyr115. This orientation suggested a distinct transition state that was substantiated from the structure of hGSTM1a-1a complexed with transition state analogue 1-S-(glutathionyl)-2,4,6-trinitrocyclohexadienate (Meisenheimer complex). Kinetic data for GSTM1a-1a indicate that kcat (CDNB) for the reaction is more than 3 times greater than k cat (FDNB), even though the nonenzymatic second-order rate constant is more than 50-fold greater for 1-fluoro-2,4-dinitrobenzene (FDNB), and the product is the same for both substrates. In addition, Km (FDNB) is about 20 times less than Km (CDNB). The results are consistent with a mechanism in which the formation of the transition state is rate-limiting in the nucleophilic aromatic substitution reactions. Data obtained with active-site mutants support transition states in which Tyr115, Tyr6, and His107 side chains are involved in the stabilization of the Meisenheimer complex via interactions with the ortho nitro group of CDNB or FDNB and provide insight into the means by which GSTs adapt to accommodate different substrates.

AB - An active site His107 residue distinguishes human glutathione S-transferase hGSTM1-1 from other mammalian Mu-class GSTs. The crystal structure of hGSTM1a-1a with bound glutathione (GSH) was solved to 1.9 Å resolution, and site-directed mutagenesis supports the conclusion that a proton transfer occurs in which bound water at the catalytic site acts as a primary proton acceptor from the GSH thiol group to transfer the proton to His107. The structure of the second substrate-binding site (H-site) was determined from hGSTM1a-1a complexed with 1-glutathionyl-2,4-dinitrobenzene (GS-DNB) formed by a reaction in the crystal between GSH and 1-chloro-2,4-dinitrobenzene (CDNB). In that structure, the GSH-binding site (G-site) is occupied by the GSH moiety of the product in the same configuration as that of the enzyme-GSH complex, and the dinitrobenzene ring is anchored between the side chains of Tyr6, Leu12, His107, Met108, and Tyr115. This orientation suggested a distinct transition state that was substantiated from the structure of hGSTM1a-1a complexed with transition state analogue 1-S-(glutathionyl)-2,4,6-trinitrocyclohexadienate (Meisenheimer complex). Kinetic data for GSTM1a-1a indicate that kcat (CDNB) for the reaction is more than 3 times greater than k cat (FDNB), even though the nonenzymatic second-order rate constant is more than 50-fold greater for 1-fluoro-2,4-dinitrobenzene (FDNB), and the product is the same for both substrates. In addition, Km (FDNB) is about 20 times less than Km (CDNB). The results are consistent with a mechanism in which the formation of the transition state is rate-limiting in the nucleophilic aromatic substitution reactions. Data obtained with active-site mutants support transition states in which Tyr115, Tyr6, and His107 side chains are involved in the stabilization of the Meisenheimer complex via interactions with the ortho nitro group of CDNB or FDNB and provide insight into the means by which GSTs adapt to accommodate different substrates.

UR - http://www.scopus.com/inward/record.url?scp=33645451885&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33645451885&partnerID=8YFLogxK

U2 - 10.1021/bi051823+

DO - 10.1021/bi051823+

M3 - Article

VL - 45

SP - 3852

EP - 3862

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 12

ER -