Formation of the 1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate anion at the active site of glutathione S-transferase: evidence for enzymic stabilization of .sigma.-complex intermediates in nucleophilic aromatic substitution reactions

Graminski, Gerard F.; Zhang, Pinghui; Sesay, Muctarr; Ammon, Herman L.; Armstrong, Richard N.

doi:10.1021/bi00441a017

Cited by 88 publications

(68 citation statements)

References 19 publications

(36 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It behaved like a suicide inhibitor: in fact, it was conjugated with GSH leading to a stable complex characterized by a single absorption band at about 350 nm. The formation of a complex or Meisenheimer complex has been observed previously in the active site of GSTs after reaction of 1,3,5-trinitrobenzene with GSH (30,31). The NBD derivatives are excellent electrophiles, readily forming complexes with many nucleophiles.…”

Section: Discussionsupporting

confidence: 57%

“…These new optical species probably represent a complex intermediate between NBDHEX and GSH stabilized in the active site of GSTP1-1 and GSTM2-2. In fact, it is well known that nucleophilic aromatic substitution reactions proceed through a complex intermediate (30,31) and that complexes of NBD have absorption bands between 300 and 400 nm (32,33). A different type of interaction was found in the presence of GSTA1-1.…”

Section: Spectrophotometric and Fluorometric Analysis At Equilibrium-mentioning

confidence: 99%

See 1 more Smart Citation

7-Nitro-2,1,3-benzoxadiazole Derivatives, a New Class of Suicide Inhibitors for Glutathione S-Transferases

Ricci¹,

Maria²,

Antonini

et al. 2005

Journal of Biological Chemistry

138

171

View full text Add to dashboard Cite

Spectroscopic and rapid kinetic experiments were performed to detail the interaction of human glutathione S-transferases GSTA1-1, GSTM2-2, and GSTP1-1 with 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX). This compound is a representative molecule of a new class of 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives (non-GSH peptidomimetic compounds) that have been designed both to give strong GST inhibition and to accumulate in tumor cells avoiding the extrusion mechanisms mediated by the multidrug resistance protein pumps. We have recently shown that submicromolar amounts of NBDHEX trigger apoptosis in several human tumor cell lines through the dissociation of the JNK⅐GSTP1-1 complex (Turella, P., Cerella, C., Filomeni, G., Bullo, A., De Maria, F., Ghibelli, L., Ciriolo, M. R., Cianfriglia, M., Mattei, M., Federici, G., Ricci, G., and Caccuri, A. M. (2005) Cancer Res. 65, 3751-3761). Results reported in the present study indicated that NBDHEX behaves like a suicide inhibitor for GSTs. It bound to the H-site and was conjugated with GSH forming a complex at the C-4 of the benzoxadiazole ring. This complex was tightly stabilized in the active site of GSTP1-1 and GSTM2-2, whereas in GSTA1-1 the release of the 6-mercapto-1-hexanol from the complex was the favored event. Docking studies demonstrated the likely localization of the complex in the GST active sites and provide a structural explanation for its strong stabilization.

show abstract

Section: Discussionsupporting

confidence: 57%

Section: Spectrophotometric and Fluorometric Analysis At Equilibrium-mentioning

confidence: 99%

7-Nitro-2,1,3-benzoxadiazole Derivatives, a New Class of Suicide Inhibitors for Glutathione S-Transferases

Ricci¹,

Maria²,

Antonini

et al. 2005

Journal of Biological Chemistry

138

171

View full text Add to dashboard Cite

show abstract

“…Crystals of these proteins were grown by vapour diffusion (for a review on protein crystallization, see [33]) and all but one [28] of the crystal forms are of enzyme complexed with a glutathione analogue as competitive inhibitor. At least two of the crystallized isoenzymes (rGSTM1-1 [34] and pGSTP1-1, Bico and Dirr, unpublished results) retain their catalytically functional conformations in the crystal lattice as demonstrated by their ability to catalyze the formation of a Meisenheimer or g-complex between glutathione and 1,3,5-trinitrobenzene in their respective protein crystals.…”

Section: Structure Analysis By X-ray Diffractionmentioning

confidence: 94%

X‐ray crystal structures of cytosolic glutathione S‐transferases

Dirr

Reinemer

Huber

1994

European Journal of Biochemistry

416

321

View full text Add to dashboard Cite

Crystal structures of cytosolic glutathione S-transferases (EC 2.5.1.18), complexed with glutathione or its analogues, are reviewed. The atomic models define protein architectural relationships between the different gene classes in the superfamily, and reveal the molecular basis for substrate binding at the two adjacent subsites of the active site. Considerable progress has been made in understanding the mechanism whereby the thiol group of glutathione is destabilized (lowering its pK,) at the active site, a rate-enhancement strategy shared by the soluble glutathione S-transferases. Portrait of a superfamily of detoxification enzymesMany sophisticated defense strategies have evolved in organisms enabling them to deal with the constant threat by a broad spectrum of both foreign and endogenous cytotoxic and genotoxic compounds [1]. Enzyme systems that transact the chemical detoxification and elimination processes of these structurally diverse molecules, many of which are highly non-polar, can be divided into three major but interrelated groups; phase I enzymes, such as the cytochrome P450 superfamily, activate chemicals by forming reactive functional groups (e.g. epoxides) in them; phase I1 enzymes deactivate these reactive chemicals by appending a hydrophilic moiety (e.g. glutathionyl, glucuronyl, or sulphuryl) to the functional group ; phase Ill enzymes mediate the cellular elimination of the inactive and water-soluble products.The superfamily of glutathione S-transferases (EC 2.5.1.18) represents an integral part of the phase I1 detoxification mechanism. These intracellular proteins are found in most aerobic eukaryotes and prokaryotes, and protect cells against chemical-induced toxicity and stress by catalyzing the S-conjugation between the thiol group of glutathione and an electrophilic moiety in the hydrophobic and toxic sub- GST, glutathione S-transferase; pGSTP1-1, hGSTAl-1, rGSTM1-1 etc., acronyms for the glutathione S-transferases (GST), the prefix indicating the species (p, porcine; h, human ; r, rat; b, bovine; m, mouse; rb, rabbit; c, chicken; gp, guinea pig) while P, A, and M indicate gene class pi, alpha and mu, respectively ; 1-1 indicates a dimer of two type-1 subunits; GSH, reduced glutathione; P1, P2 etc. and G1, G2 etc., designate peptide functional groups in glutathione and the corresponding G-site ligands of the glutathione S-transferases, respectively; G-site, glutatbione-binding site; H-site, hydrophobic electrophile-binding site.Enzyme. Glutathione S-transferase (EC 2.5.1.1 8).strate. Glutathione S-transferases are perhaps the single most important family of enzymes involved in the metabolism of alkylating compounds [2]. The resultant glutathione S-conjugates can be exported from animal cells by putative membrane ATP-dependent pump systems [3] after which they are metabolized via the mercapturate pathway and eventually eliminated. An extraordinary feature of the glutathione S-transferase superfamily is the occurrence of multiple enzyme forms that, according to sequence similarities and subc...

show abstract

“…Independent evidence for the intermediacy of EMc derives from a stoppedflow absorption kinetic analysis of the dehalogenase-catalyzed reactions of 4-CBA-CoA analogues (12). These findings, coupled with "chemical logic" (there is ample evidence in the literature for stepwise nucleophilic substitution on activated aromatic rings but none supporting a concerted mechanism) (24)(25)(26)(27), are most consistent with the two-covalent intermediate kinetic model of Scheme 2, and thus support its use in the determination of the microscopic rate constants. 14 Clabeled enzyme.…”

Section: X-ray Crystal Structure Of the H90q Dehalogenase-4-hba-coa Cmentioning

confidence: 99%

Histidine 90 Function in 4-Chlorobenzoyl-Coenzyme A Dehalogenase Catalysis^,

Zhang

Wei²,

Luo³

et al. 2001

Biochemistry

View full text Add to dashboard Cite

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA by attack of Asp145 on the C(4) of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of chloride ion to form an arylated enzyme intermediate (EAr) and, finally, ester hydrolysis in EAr to form 4-hydroxybenzoyl-CoA (4-HBA-CoA). This study examines the contribution of the active site His90 to catalysis of this reaction pathway. The His90 residue was replaced with glutamine by site-directed mutagenesis. X-ray crystallographic analysis of H90Q dehalogenase complexed with 4-HBA-CoA revealed that the positions of the catalytic groups are unchanged from those observed in the structure of the 4-HBA-CoA-wild-type dehalogenase complex. The one exception is the Gln90 side chain, which is rotated away from the position of the His90 side chain. The vacated His90 site is occupied by two water molecules. Kinetic techniques were used to evaluate ligand binding and catalytic turnover rates in the wild-type and H90Q mutant dehalogenases. The rate constants for 4-CBA-CoA (both 7 µM -1 s -1 ) and 4-HBA-CoA (33 and 11 µM -1 s -1 ) binding to the two dehalogenases are similar in value. For wild-type dehalogenase, the rate constant for a single turnover is 2.3 s -1 while that for multiple turnovers is 0.7 s -1 . For H90Q dehalogenase, these rate constants are 1.6 × 10 -2 and 2 × 10 -4 s -1 . The rate constants for EMc formation in wild-type and mutant dehalogenase are ∼200 s -1 while the rate constants for EAr formation are 40 and 0.3 s -1 , respectively. The rate constant for hydrolysis of EAr in wild-type dehalogenase is 20 s -1 and in the H90Q mutant, 0.13 s -1 . The 133-fold reduction in the rate of EAr formation in the mutant may be the result of active site hydration, while the 154-fold reduction in the rate EAr hydrolysis may be the result of lost general base catalysis. Substitution of the His90 with Gln also introduces a rate-limiting step which follows catalysis, and may involve renewing the catalytic site through a slow conformational change.

show abstract

Formation of the 1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate anion at the active site of glutathione S-transferase: evidence for enzymic stabilization of .sigma.-complex intermediates in nucleophilic aromatic substitution reactions

Cited by 88 publications

References 19 publications

7-Nitro-2,1,3-benzoxadiazole Derivatives, a New Class of Suicide Inhibitors for Glutathione S-Transferases

7-Nitro-2,1,3-benzoxadiazole Derivatives, a New Class of Suicide Inhibitors for Glutathione S-Transferases

X‐ray crystal structures of cytosolic glutathione S‐transferases

Histidine 90 Function in 4-Chlorobenzoyl-Coenzyme A Dehalogenase Catalysis^,

Contact Info

Product

Resources

About

Formation of the 1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate anion at the active site of glutathione S-transferase: evidence for enzymic stabilization of .sigma.-complex intermediates in nucleophilic aromatic substitution reactions

Cited by 88 publications

References 19 publications

7-Nitro-2,1,3-benzoxadiazole Derivatives, a New Class of Suicide Inhibitors for Glutathione S-Transferases

7-Nitro-2,1,3-benzoxadiazole Derivatives, a New Class of Suicide Inhibitors for Glutathione S-Transferases

X‐ray crystal structures of cytosolic glutathione S‐transferases

Histidine 90 Function in 4-Chlorobenzoyl-Coenzyme A Dehalogenase Catalysis,

Contact Info

Product

Resources

About

Histidine 90 Function in 4-Chlorobenzoyl-Coenzyme A Dehalogenase Catalysis^,