Structure and reaction mechanism of human nicotinamide phosphoribosyltransferase.
Structure and reaction mechanism of human nicotinamide phosphoribosyltransferase.
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DOI：
10.1093/jb/mvp152
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发表时间：
2010
影响因子：
2.7
通讯作者：
R. Takahashi;S. Nakamura;T. Nakazawa;K. Minoura;Takuya Yoshida;Y. Nishi;Yuji Kobayashi;T. Ohkubo
中科院分区：
文献类型：
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作者：
R. Takahashi;S. Nakamura;T. Nakazawa;K. Minoura;Takuya Yoshida;Y. Nishi;Yuji Kobayashi;T. Ohkubo
烟酰胺（NM）磷酸核糖转移酶（NMPRTase）在NAD生物合成途径中催化NM与5 '-磷酸核糖-1'-焦磷酸（PRPP）反应生成NM单核苷酸（NMN）和焦磷酸（PPi）。监测反应混合物的（1）H和（31）P NMR谱，我们发现该反应是可逆的，如平衡常数K = [NMN][PPi]/（[NM][PRPP]）= 0.14所指示的，其与正向和反向反应的二级速率常数的比率K = 0.16很好地一致。该酶以游离形式结合到NM和PRPP上的晶体结构在2.0-2.2 A的分辨率下与NMN复合物的晶体结构基本上相同，除了一些变化，这些变化可以通过固定亲核试剂和离去基团来促进取代反应，从而在核糖环的C1'碳上进行必要的构型反转。在结合的PRPP或NMN的C1'原子附近的活性位点中，既没有带负电荷的基团，也没有支持S（N）1机制中固有的核糖-氧代碳正离子中间体的可行性所必需的防水环境。本文还结合NMPRTase的多种生物学功能对NMPRTase的结构和催化机理进行了讨论。
Nicotinamide (NM) phosphoribosyltransferase (NMPRTase) catalyzes the reaction of NM and 5'-phosphoribosyl-1'-pyrophosphate (PRPP) to form NM mononucleotide (NMN) and pyrophosphate (PPi) in the pathway of NAD-biosynthesis. Monitoring the (1)H and (31)P 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 A 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 S(N)1 mechanism. The structures and catalytic mechanism thus revealed are also discussed in connection with the multiple biological functions of NMPRTase.