Weak Coupling of ATP Hydrolysis to the Chemical Equilibrium of Human Nicotinamide Phosphoribosyltransferase

Abstract

Human nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) catalyses the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PPi) from nicotinamide (NAM) and α-D-5-phosphoribosyl-1-pyrophosphate (PRPP). NAMPT, by capturing the energy provided by its facultative ATPase activity, allows the production of NMN at products/ substrates ratios thermodynamically forbidden in the absence of ATP. By coupling ATP hydrolysis to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold and substrate affinity dramatically increased (K m NAM from 855 nM to 5 nM) and the K eq shifted −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 catalysis 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 non optimized 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 µM) and NADH (K i = 0.22 µM), an apparent regulatory feedback mechanism.The properties of NAD + in electron transfer are now established. As a cofactor in many metabolic pathways, NAD + holds a key position in energy metabolism and its regulation. Over the last decade, new involvements for NAD + were discovered. It is used by poly and mono (ADP-ribose) polymerases as a substrate for protein covalent modifications (1-5).Sirtuins (SIRT) use NAD + in protein deacetylation reactions with implications for metabolic control, cancer and longevity. While redox reactions do not influence the net level of NAD + + NADH concentration, enzymes that catalyze ADP-ribosylation and deacetylation cleave the N-ribosyl bond to nicotinamide and can lead to a rapid depletion of this substrate. NAD + levels influence all of its associated pathways and the NAD + pool needs constant replenishment from NAM recycling since dietary sources of the vitamin may be limiting.In prokaryotes and lower eukaryotes, NAD + replenishment is achieved primarily by de novo synthesis. Quinolinic acid, derived from tryptophan, is converted into nicotinic acid mononucleotide (NAMN) by the quinolinic acid phosphoribosyltransferase (QAPT). NAMN, obtained from exogenous nicotinic acid (NA) by reaction with nicotinic acid phosphoribosyltransferase (NAPT), is converted into NAD + , via the Preiss-Handler pathway † This work was supported by research grant GM41916 from the NIH. *To whom correspondence should be addressed at the Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel: (718) 430-281...

A phosphoenzyme mimic, overlapping catalytic sites and reaction coordinate motion for human NAMPT

“…The activation of NAMPT by ATP hydrolysis has suggested that phosphorylation on a conserved histidine is a common catalytic feature for the pyridine phosphoribosyltransferases (6,11,15). In the Fig.…”

“…presence of Mg 2ϩ and ATP, NAMPT undergoes a fast, near quantitative phosphorylation (6). Phospho-NAMPT is approximately 3 orders of magnitude more active than unphosphorylated enzyme.…”

“…Nicotinamide phosphoribosyltransferase (NAMPT; also known as pre-␤ cell colony enhancing factor, PBEF, and visfatin, an adipokine) is designed to efficiently recycle NAM (K m ϭ 5 nM) by reaction with ␣-D-5-phosphoribosyl-1-pyrophosphate (PRPP, Fig. 1) to sustain the pool of NAD ϩ (6).…”

“…Both transferases have an ATPase activity that nonstoichiometrically couples with the transferase reaction to shift the thermodynamic equilibrium toward mononucleotide formation (6,12). For Salmonella typhimurium NAPT (13,14) the ATPase reaction involves phosphorylation of H219.…”

“…The NAMPT inhibitor, FK866 (10) has validated the enzyme as a target for the development of new anticancer agents. Although it binds with high picomolar affinity, the K m /K i is only 33, because the enzyme has an extraordinary affinity for NAM (K m NAM of 5 nM) when it is in its phosphorylated state (6). The unusual catalytic site of human NAMPT (11) [supporting information (SI) Fig.…”