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

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

Research output: Contribution to journalArticle

23 Citations (Scopus)

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

Fingerprint

Niacinamide
Nicotinamide Mononucleotide
Recycling
Isotopes
Nucleophiles
Kinetics
Catalytic Domain
Enzymes
NAD+ Nucleosidase
Sirtuins
Nicotinamide Phosphoribosyltransferase
Administrative data processing
Poly(ADP-ribose) Polymerases
NAD
Torsional stress
Magnesium
Crystal structure
Atoms
Molecules
Water

ASJC Scopus subject areas

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

Cite this

Recycling nicotinamide. the transition-state structure of human nicotinamide phosphoribosyltransferase. / Burgos, Emmanuel S.; Vetticatt, Mathew J.; Schramm, Vern L.

In: Journal of the American Chemical Society, Vol. 135, No. 9, 06.03.2013, p. 3485-3493.

Research output: Contribution to journalArticle

@article{9a5439992b104db3a672590ace8bce7c,
title = "Recycling nicotinamide. the transition-state structure of human nicotinamide phosphoribosyltransferase",
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 {\AA}), and the N-ribosidic C1′-N bond is highly elongated at the TS (rC-N = 2.35 {\AA}), 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 {\AA} migration of C1′.",
author = "Burgos, {Emmanuel S.} and Vetticatt, {Mathew J.} and Schramm, {Vern L.}",
year = "2013",
month = "3",
day = "6",
doi = "10.1021/ja310180c",
language = "English (US)",
volume = "135",
pages = "3485--3493",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "9",

}

TY - JOUR

T1 - Recycling nicotinamide. the transition-state structure of human nicotinamide phosphoribosyltransferase

AU - Burgos, Emmanuel S.

AU - Vetticatt, Mathew J.

AU - Schramm, Vern L.

PY - 2013/3/6

Y1 - 2013/3/6

N2 - 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′.

AB - 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′.

UR - http://www.scopus.com/inward/record.url?scp=84874908355&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84874908355&partnerID=8YFLogxK

U2 - 10.1021/ja310180c

DO - 10.1021/ja310180c

M3 - Article

C2 - 23373462

AN - SCOPUS:84874908355

VL - 135

SP - 3485

EP - 3493

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 9

ER -