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Journal of Biochemistry Advance Access published online on October 9, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp152
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Structure and Reaction Mechanism of Human Nicotinamide Phosphoribosyltransferase

Ryo Takahashi1, Shota Nakamura1, Takashi Nakazawa2, Katsuhiko Minoura3, Takuya Yoshida1, Yoshinori Nishi3, Yuji Kobayashi3 and Tadayasu Ohkubo1

1 Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan, 2 Department of Chemistry, Nara Women's University, Nara 630-8506, Japan, 3 Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan.

Corresponding author: Yuji Kobayashi, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan, Telephone/Fax: +81-72-690-1080, E-mail: kobayasi{at}gly.oups.ac.jp

Received August 26, 2009; Accepted September 2, 2009


  Abstract

Nicotinamide phosphoribosyltransferase (NMPRTase) catalyzes the reaction of nicotinamide (NM) and 5'-phosphoribosyl-1'-pyrophosphate (PRPP) to form nicotinamide mononucleotide (NMN) and pyrophosphate (PPi) in the pathway of NAD-biosynthesis. Monitoring the 1H and 31P NMR spectra of the reaction mixture, we found that this reaction is reversible as dictated by the equilibrium constant K = [NMN][PPi]/([NM][PRPP]) = 0.14, which agreed well with the ratio of second-order rate constants for forward and backward reactions, K = 0.16. The crystal structures of this enzyme in the free form and bound to NM and PRPP at the resolution of 2.0 – 2.2 Å were essentially identical to that of the complex with NMN, except for some variations that could facilitate the substitution reaction by fixing the nucleophile and the leaving group for the requisite inversion of configuration at the C1' carbon of the ribose ring. In the active site near the C1' atom of the bound PRPP or NMN, there was neither negatively charged group nor waterproof environment necessary to support the feasibility of a ribo-oxocarbocation intermediate inherent in the SN1 mechanism. The structures and catalytic mechanism thus revealed are also discussed in connection with the multiple biological functions of NMPRTase.

Key Words: chemical equilibrium, multifunctional enzyme, nicotinamide phosphoribosyltransferase, SN2, visfatin, X-ray analysis


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