Kinetic studies and active site-binding properties of glutathione S-transferase using spin-labeled glutathione, a product analogue

Vern L. Schramm, R. McCluskey, F. A. Emig, G. Litwack

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

Kinetic and binding studies with substrates, products, and a spin-labeled product analogue of glutathione (sl-glutathione) have been used to characterize the kinetic mechanism and properties of the catalytic site of the homodimer Y(a)Y(a) of glutathione S-transferase. Product inhibition studies and inhibition by sl-glutathione indicate the random addition of substrates. The kinetically determined dissociation constant for the product S-(2,4-dinitrophenyl)glutathione is approximately 7 μM. A newly described spin-labeled product analogue, S-[[(2,2,5,5,-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl]glutathio ne (sl-glutathione), acts as a competitive inhibitor with respect to both substrates (glutathione and 1-Cl-2,4-dinitrobenzene) with a kinetically determined dissociation constant of approximately 40 μM. Analysis of the glutathione S-transferase/sl-glutathione complex by EPR gives a rigid limit spectrum indicative of highly immobilized spin label. Kinetic and EPR results support the proposal that sl-glutathione binds as a bisubstrate or product analogue by occupying both the gluthathione and hydrophobic substrate sites. Binding studies of sl-glutathione by EPR give a dissociation constant of 28 μM and a single binding site per homodimer. Displacement of sl-glutathione by substrates and product have been used to directly determine enzyme-ligand dissociation constants. Dissociation constants of 2.1 mM, 17 μM, and 25 μM were obtained for glutathione, 1-Cl-2.4-dinitrobenzene and S-(2,4-dinitrophenyl)glutathione when enzyme was added to a mixture of sl-glutathione and the competing ligand. The dissociation constants for glutathione and 1-Cl-2,4-dinitrobenzene but not for S-(2,4-dinitrophenyl)glutathione were dependent on the order of addition, consistent with the existence of several kinetically stable conformations for the enzyme. The sl-glutathione described here may provide a useful analogue for similar studies with other glutathione S-transferases or other enzymes which bind glutathione.

Original languageEnglish (US)
Pages (from-to)714-722
Number of pages9
JournalJournal of Biological Chemistry
Volume259
Issue number2
StatePublished - 1984
Externally publishedYes

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Glutathione Transferase
Glutathione
Catalytic Domain
Binding Sites
Kinetics
Paramagnetic resonance
Substrates
Enzymes
Dinitrobenzenes
Ligands
Spin Labels
Conformations

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic studies and active site-binding properties of glutathione S-transferase using spin-labeled glutathione, a product analogue. / Schramm, Vern L.; McCluskey, R.; Emig, F. A.; Litwack, G.

In: Journal of Biological Chemistry, Vol. 259, No. 2, 1984, p. 714-722.

Research output: Contribution to journalArticle

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abstract = "Kinetic and binding studies with substrates, products, and a spin-labeled product analogue of glutathione (sl-glutathione) have been used to characterize the kinetic mechanism and properties of the catalytic site of the homodimer Y(a)Y(a) of glutathione S-transferase. Product inhibition studies and inhibition by sl-glutathione indicate the random addition of substrates. The kinetically determined dissociation constant for the product S-(2,4-dinitrophenyl)glutathione is approximately 7 μM. A newly described spin-labeled product analogue, S-[[(2,2,5,5,-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl]glutathio ne (sl-glutathione), acts as a competitive inhibitor with respect to both substrates (glutathione and 1-Cl-2,4-dinitrobenzene) with a kinetically determined dissociation constant of approximately 40 μM. Analysis of the glutathione S-transferase/sl-glutathione complex by EPR gives a rigid limit spectrum indicative of highly immobilized spin label. Kinetic and EPR results support the proposal that sl-glutathione binds as a bisubstrate or product analogue by occupying both the gluthathione and hydrophobic substrate sites. Binding studies of sl-glutathione by EPR give a dissociation constant of 28 μM and a single binding site per homodimer. Displacement of sl-glutathione by substrates and product have been used to directly determine enzyme-ligand dissociation constants. Dissociation constants of 2.1 mM, 17 μM, and 25 μM were obtained for glutathione, 1-Cl-2.4-dinitrobenzene and S-(2,4-dinitrophenyl)glutathione when enzyme was added to a mixture of sl-glutathione and the competing ligand. The dissociation constants for glutathione and 1-Cl-2,4-dinitrobenzene but not for S-(2,4-dinitrophenyl)glutathione were dependent on the order of addition, consistent with the existence of several kinetically stable conformations for the enzyme. The sl-glutathione described here may provide a useful analogue for similar studies with other glutathione S-transferases or other enzymes which bind glutathione.",
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