Glutathione reductase: solvent equilibrium and kinetic isotope effects
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 microM and 13 microM, 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.
- Research Organization:
- Albert Einstein College of Medicine, Bronx, NY (USA)
- OSTI ID:
- 6786631
- Journal Information:
- Biochemistry; (United States), Vol. 27:18
- Country of Publication:
- United States
- Language:
- English
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DEUTERIUM
ISOTOPE EFFECTS
OXIDOREDUCTASES
BIOCHEMICAL REACTION KINETICS
DISULFIDES
GLUTATHIONE
IN VITRO
NADP
PROTONS
SUBSTRATES
TRACER TECHNIQUES
BARYONS
COENZYMES
DRUGS
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
HADRONS
HYDROGEN ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
NUCLEI
NUCLEONS
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PEPTIDES
POLYPEPTIDES
PROTEINS
RADIOPROTECTIVE SUBSTANCES
REACTION KINETICS
STABLE ISOTOPES
550201* - Biochemistry- Tracer Techniques