Recycling nicotinamide. the transition-state structure of human nicotinamide phosphoribosyltransferase

Emmanuel S. Burgos, Mathew J. Vetticatt, Vern L. Schramm

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Abstract

Human nicotinamide phosphoribosyltransferase (NAMPT) replenishes the NAD pool and controls the activities of sirtuins, mono- and poly-(ADP-ribose) polymerases, and NAD nucleosidase. The nature of the enzymatic transition-state (TS) is central to understanding the function of NAMPT. We determined the TS structure for pyrophosphorolysis of nicotinamide mononucleotide (NMN) from kinetic isotope effects (KIEs). With the natural substrates, NMN and pyrophosphate (PPi), the intrinsic KIEs of [1′-14C], [1- 15N], [1′-3H], and [2′-3H] are 1.047, 1.029, 1.154, and 1.093, respectively. A unique quantum computational approach was used for TS analysis that included structural elements of the catalytic site. Without constraints (e.g., imposed torsion angles), the theoretical and experimental data are in good agreement. The quantum-mechanical calculations incorporated a crucial catalytic site residue (D313), two magnesium atoms, and coordinated water molecules. The TS model predicts primary 14C, α-secondary 3H, β-secondary 3H, and primary 15N KIEs close to the experimental values. The analysis reveals significant ribocation character at the TS. The attacking PPi nucleophile is weakly interacting (rC-O = 2.60 Å), and the N-ribosidic C1′-N bond is highly elongated at the TS (rC-N = 2.35 Å), consistent with an ANDN mechanism. Together with the crystal structure of the NMN·PPi·Mg2· enzyme complex, the reaction coordinate is defined. The enzyme holds the nucleophile and leaving group in relatively fixed positions to create a reaction coordinate with C1′-anomeric migration from NAM to the PPi. The TS is reached by a 0.85 Å migration of C1′.

Original languageEnglish (US)
Pages (from-to)3485-3493
Number of pages9
JournalJournal of the American Chemical Society
Volume135
Issue number9
DOIs
StatePublished - Mar 6 2013

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ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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