Recycling nicotinamide. The transition-state structure of human nicotinamide phosphoribosyltransferase.
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'-(14)C], [1-(15)N], [1'-(3)H], and [2'-(3)H] 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 (14)C, α-secondary (3)H, β-secondary (3)H, and primary (15)N KIEs close to the experimental values. The analysis reveals significant ribocation character at the TS. The attacking PPi nucleophile is weakly interacting (r(C-O) = 2.60 Å), and the N-ribosidic C1'-N bond is highly elongated at the TS (r(C-N) = 2.35 Å), consistent with an A(N)D(N) 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'.
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, United States.
Emmanuel S
ES
Burgos
Mathew J
MJ
Vetticatt
Vern L
VL
Schramm
D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D015394:Molecular Structure; D009536:Niacinamide / Q000096:biosynthesis* / Q000737:chemistry; D054409:Nicotinamide Phosphoribosyltransferase / Q000737:chemistry / Q000378:metabolism*
D009536:Niacinamide; D054409:Nicotinamide Phosphoribosyltransferase