Muller-Steffner H M, Augustin A, Schuber F
Laboratoire de Chimie Bioorganique, Laboratoire Associé au Centre National de la Recherche Scientifique 1386, Faculté de Pharmacie, Université Louis Pasteur Strasbourg, 74 route du Rhin, 67400 Illkirch, France.
J Biol Chem. 1996 Sep 27;271(39):23967-72. doi: 10.1074/jbc.271.39.23967.
We have shown that bovine spleen NAD+ glycohydrolase (EC), purified to homogeneity, is a multifunctional enzyme. A time-dependent formation of cADPR from NAD+ that did not exceed 1.5-2% of the reaction products was measurable. The cyclase activity of this enzyme was, however, best evidenced by its transformation of NGD+ into cyclic GDP-ribose (cGDPR). The formation of the cyclic compound could be monitored spectroscopically (UV and fluorescence) and by high-performance liquid chromatography; the product ratio of cGDPR/GDP-ribose was 2:1. Bovine spleen NAD+ glycohydrolase is also able to hydrolyze cADPR (Muller-Steffner et al. (1994) Biochem. Biophys. Res. Commun. 204, 1279-1285); the kinetic parameters (V/Km) measured exclude, however, the possibility that cADPR is a kinetically competent reaction intermediate in the transformation of NAD+ into ADP-ribose. Experimental data indicating that NAD+ glycohydrolase-catalyzed hydrolysis and methanolysis of NA(G)D+ occurred at the expense of the formation of the cyclic compounds are in favor of a reaction mechanism involving the partitioning of a common oxocarbenium reaction intermediate between the different acceptors. Thus E.A(G)DP-ribosyl oxocarbenium intermediate can react according to i) intramolecular processes with the positions N-1 of adenine and N-7 of guanine to give cA(G)DPR as reaction products, and ii) intermolecular reactions with water (formation of A(G)DP-ribose) and methanol (formation of methyl A(G)DP-ribose). We attribute the marked difference in yield of cADPR and cGDPR to the intrinsic reactivity (nucleophilicity and positioning) of the purine N-positions that are involved in the cyclization reactions within the E.A(G)DP-ribosyl oxocarbenium complexes.
我们已经证明，纯化至同质的牛脾NAD⁺糖水解酶（EC）是一种多功能酶。可检测到由NAD⁺随时间形成的cADPR，其产量不超过反应产物的1.5 - 2%。然而，该酶的环化酶活性最有力的证据是其将NGD⁺转化为环鸟苷二磷酸核糖（cGDPR）。环状化合物的形成可以通过光谱法（紫外和荧光）以及高效液相色谱法进行监测；cGDPR/鸟苷二磷酸核糖的产物比例为2:1。牛脾NAD⁺糖水解酶也能够水解cADPR（Muller - Steffner等人，（1994年）《生物化学与生物物理研究通讯》204，1279 - 1285）；然而，所测量的动力学参数（V/Km）排除了cADPR是NAD⁺转化为ADP - 核糖过程中动力学上合适的反应中间体的可能性。实验数据表明，NAD⁺糖水解酶催化的NA(G)D⁺水解和甲醇解是以环状化合物的形成为代价发生的，这支持了一种反应机制，即涉及一个共同的氧碳鎓反应中间体在不同受体之间的分配。因此，E.A(G)DP - 核糖基氧碳鎓中间体可以根据以下方式反应：i）与腺嘌呤的N - 1位和鸟嘌呤的N - 7位进行分子内反应，生成cA(G)DPR作为反应产物；ii）与水（形成A(G)DP - 核糖）和甲醇（形成甲基A(G)DP - 核糖）进行分子间反应。我们将cADPR和cGDPR产量的显著差异归因于E.A(G)DP - 核糖基氧碳鎓复合物中环化反应所涉及的嘌呤N位的固有反应性（亲核性和定位）。
