Fig. 1.

Human NAMPT exists in P-H247 (red) and H247 (blue) forms. P-H247 NAMPT is 1,125-fold more active (k_cat/K_m values) and requires continual hydrolysis of ATP. P-H247 NAMPT has low K_m values for NAM and PRPP (red) while H247 NAMPT has higher K_m values (blue). NAMPT slowly hydrolyzes ATP (orange) even in the absence of NAM and PRPP. In the presence of ATP, the stoichiometry of NMN:ADP formation is always 1.0 or less, indicating P-H247 hydrolysis on each NAM synthetic cycle. PRPP has a tighter affinity than NAM for P-H247, suggesting a preferential ordered binding of substrates to the enzyme, with PRPP binding first. Five crystallographic structures illustrate the main steps of this complex mechanism.