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

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