Journal
BIOCHEMISTRY
Volume 47, Issue 42, Pages 11086-11096Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi801198m
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Funding
- National Institutes of Health [GM41916]
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Human nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) catalyzes the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PP(i)) from nicotinamide (NAM) and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). NAMPT, by capturing the energy provided by its facultative ATPase activity, allows the production of NMN at product: substrate ratios thermodynamically forbidden in the absence of ATP. With ATP hydrolysis coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, substrate affinity dramatically increases (K(m)(NAM) from 855 to 5 nM), and the K(eq) shifts -2.1 kcal/mol toward NMN formation. ADP-ATP isotopic exchange experiments support the formation of a high-energy phosphorylated intermediate (phospho-H247) as the mechanism for altered catalytic efficiency during ATP hydrolysis. NAMPT captures only a small portion of the energy generated by ATP hydrolysis to shift the dynamic chemical equilibrium. Although the weak energetic coupling of ATP hydrolysis appears to be a nonoptimized enzymatic function, closer analysis of this remarkable protein reveals an enzyme designed to capture NAM with high efficiency at the expense of ATP hydrolysis. NMN is a rate-limiting precursor for recycling to the essential regulatory cofactor, nicotinamide adenine dinucleotide (NAD(+)). NMN synthesis by NAMPT is powerfully inhibited by both NAD(+) (K(i) = 0.14 mu M) and NADH (K(i) = 0.22 mu M), an apparent regulatory feedback mechanism.
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