Kinetic parameters in the NAMPT and NAPRT reactions.

NAMPT (36 ng) was incubated with various Nam concentrations at a fixed PRPP concentration (300 µM) for 7 and 20 min in the presence or absence of 1 mM ATP, respectively. The enzyme (7 and 36 ng) was also incubated with various concentrations of PRPP at fixed Nam concentrations (5 and 40 µM) for 7 and 30 min in the presence or absence of 1 mM ATP, respectively. NAPRT (70 and 100 ng) was incubated with various NA concentrations at a fixed PRPP concentration (300 µM) in the presence or absence of 1 mM ATP, respectively, for 30 min. The enzyme (12 and 90 ng) was also incubated with various concentrations of PRPP at fixed NA concentrations (50 and 380 µM) for 15 and 30 min in the presence or absence of 1 mM ATP, respectively. The mutant NAPRT (H213N-NAPRT, 140 ng) was incubated with various NA concentrations at a fixed PRPP concentration (600 µM) or with various concentrations of PRPP at a fixed NA concentration (650 µM) in the presence or absence of 1 mM ATP for 60 min. Km and Vmax values represent the mean ± S.D. of at least three separate experiments.