Glutathione reductase: Solvent equilibrium and kinetic isotope effects

Kenny K. Wong, John S. Blanchard, John S. Blanchard

Research output: Contribution to journalArticle

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Abstract

Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D2O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 μM and 13 μM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456.

Original languageEnglish (US)
Pages (from-to)7091-7096
Number of pages6
JournalBiochemistry
Volume27
Issue number18
StatePublished - 1988

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Glutathione Reductase
Isotopes
Glutathione Disulfide
Kinetics
Protons
Proton transfer
Yeast
Substrates
Spinacia oleracea
Yeasts
Erythrocytes
NADP
Dithionitrobenzoic Acid
Equipment and Supplies
Fractionation
Sulfhydryl Compounds
Disulfides
Enzymes
Chemical analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Glutathione reductase : Solvent equilibrium and kinetic isotope effects. / Wong, Kenny K.; Blanchard, John S.; Blanchard, John S.

In: Biochemistry, Vol. 27, No. 18, 1988, p. 7091-7096.

Research output: Contribution to journalArticle

Wong, KK, Blanchard, JS & Blanchard, JS 1988, 'Glutathione reductase: Solvent equilibrium and kinetic isotope effects', Biochemistry, vol. 27, no. 18, pp. 7091-7096.
Wong, Kenny K. ; Blanchard, John S. ; Blanchard, John S. / Glutathione reductase : Solvent equilibrium and kinetic isotope effects. In: Biochemistry. 1988 ; Vol. 27, No. 18. pp. 7091-7096.
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