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.(ABSTRACT TRUNCATED AT 250 WORDS)