Glutathione reductase: Comparison of steady-state and rapid reaction primary kinetic isotope effects exhibited by the yeast, spinach, and Escherichia coli enzymes

Journal Article · Tue Jun 19 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00476a021· OSTI ID:6795416

Vanoni, M A; Wong, K K; Ballou, D P; Blanchard, J S ^[1]

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1. Albert Einstein College of Medicine, Bronx, NY (USA)

Kinetic parameters for NADPH and NADH have been determined at pH 8.1 for spinach, yeast, and E. coli glutathione reductases. NADPH exhibited low Km values for all enzymes (3-6 microM), while the Km values for NADH were 100 times higher (approximately 400 microM). Under our experimental conditions, the percentage of maximal velocities with NADH versus those measured with NADPH were 18.4, 3.7, and 0.13% for the spinach, yeast, and E. coli enzymes, respectively. Primary deuterium kinetic isotope effects were independent of GSSG concentration between Km and 15Km levels, supporting a ping-pong kinetic mechanism. For each of the three enzymes, NADPH yielded primary deuterium kinetic isotope effects on Vmax only, while NADH exhibited primary deuterium kinetic isotope effects on both V and V/K. The magnitude of DV/KNADH at pH 8.1 is 4.3 for the spinach enzyme, 2.7 for the yeast enzyme, and 1.6 for the E. coli glutathione reductase. The experimentally determined values of TV/KNADH of 7.4, 4.2, and 2.2 for the spinach, yeast, and E. coli glutathione reductases agree well with those calculated from the corresponding DV/KNADH using the Swain-Schaad expression. This suggests that the intrinsic primary kinetic isotope effect on NADH oxidation is fully expressed. In order to confirm this conclusion, single-turnover experiments have been performed. The measured primary deuterium kinetic isotope effects on the enzyme reduction half-reaction using NADH match those measured in the steady state for each of the three glutathione reductases.

• OSTI ID: 6795416
• Journal Information: Biochemistry; (USA), Vol. 29:24; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DEUTERIUM
ISOTOPE EFFECTS
OXIDOREDUCTASES
BIOCHEMICAL REACTION KINETICS
COMPARATIVE EVALUATIONS
ESCHERICHIA COLI
GLUTATHIONE
NADH2
NADP
SPINACH
STEADY-STATE CONDITIONS
TRACER TECHNIQUES
YEASTS
BACTERIA
COENZYMES
DRUGS
ENZYMES
EUMYCOTA
FOOD
FUNGI
HYDROGEN ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAGNOLIOPHYTA
MAGNOLIOPSIDA
MICROORGANISMS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PEPTIDES
PLANTS
POLYPEPTIDES
PROTEINS
RADIOPROTECTIVE SUBSTANCES
REACTION KINETICS
STABLE ISOTOPES
VEGETABLES
550201* - Biochemistry- Tracer Techniques