A Phosphoenzyme Mimic, Overlapping Catalytic Sites and Reaction Coordinate Motion for Human NAMPT

Nicotinamide phosphoribosyltransferase (NAMPT) is highly evolved to capture nicotinamide (NAM) and replenish the nicotinamide adenine dinucleotide (NAD⁺) pool during ADP-ribosylation and transferase reactions. ATP-phosphorylation of an active-site histidine causes catalytic activation, increasing NA...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2009-08, Vol.106 (33), p.13748-13753
Main Authors: Burgos, Emmanuel S., Ho, Meng-Chiao, Almo, Steven C., Schramm, Vern L., Walsh, Christopher T.
Format: Article
Language:English
Subjects:
Active sites
Adenosine triphosphatase
Adenosine triphosphatases
Adenosine Triphosphatases - chemistry
Adenosine Triphosphate - chemistry
Atoms
Binding Sites
Biochemistry
Biological Sciences
Catalysis
Catalytic Domain
Coordinate systems
Crystallization
Crystallography, X-Ray - methods
Crystals
Diphosphates
Diphosphates - chemistry
Enzyme Activation
Enzyme kinetics
Enzymes
Histidine - chemistry
Humans
Hydrogen bonds
Kinetics
Motion
Niacinamide - chemistry
Nicotinamide Phosphoribosyltransferase - chemistry
Phosphorylation
Reaction kinetics
Substrate Specificity
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Summary:Nicotinamide phosphoribosyltransferase (NAMPT) is highly evolved to capture nicotinamide (NAM) and replenish the nicotinamide adenine dinucleotide (NAD⁺) pool during ADP-ribosylation and transferase reactions. ATP-phosphorylation of an active-site histidine causes catalytic activation, increasing NAM affinity by 160,000. Crystal structures of NAMPT with catalytic site ligands identify the phosphorylation site, establish its role in catalysis, demonstrate unique overlapping ATP and phosphoribosyltransferase sites, and establish reaction coordinate motion. NAMPT structures with beryllium fluoride indicate a covalent H247-BeF₃⁻ as the phosphohistidine mimic. Activation of NAMPT by H247-phosphorylation causes stabilization of the enzyme-phosphoribosylpyrophosphate complex, permitting efficient capture of NAM. Reactant and product structures establish reaction coordinate motion for NAMPT to be migration of the ribosyl anomeric carbon from the pyrophosphate leaving group to the nicotinamide-NI while the 5-phosphoryl group, the pyrophosphate moiety, and the nicotinamide ring remain fixed in the catalytic site.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0903898106