Kinetic parameters for NADPH and NADH have been determined at pH 8.1 for spinach, yeast, and E. coli glutathione reductases. NADPH exhibited low K(m) values for all enzymes (3-6 μM), while the K(m) values for NADH were 100 times higher (~400 μM). 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 K(m) and 15K(m) levels, supporting a ping-pong kinetic mechanism. For each of the three enzymes, NADPH yielded primary deuterium kinetic isotope effects on V(max) only, while NADH exhibited primary deuterium kinetic isotope effects on both V and V/K. The magnitude of (D)V/K(NADH) 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 redutase. The experimentally determined values of (T)V/K(NADH) 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 (D)V/K(NADH) 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-reduction using NADH match those measured in the steady state for each of the three glutathione reductases. These data support the conclusions that NADH dissociates from the binary E-NADH complex more rapidly than it transfers a hydride ion to flavin and that with NADH, intrinsic primary deuterium kinetic isotope effects are observed for all three enzymes. The substantial differences in the magnitudes of the intrinsic deuterium kinetic isotope effects on hydride transfer may reflect differences in the transition-state structures for the hydride-transfer step catalyzed by the three enzymes.
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