Sialic acid biosynthesis:: Stereochemistry and mechanism of the reaction catalyzed by the mammalian UDP-N-acetylglucosamine 2-epimerase
Sialic acid biosynthesis:: Stereochemistry and mechanism of the reaction catalyzed by the mammalian UDP-N-acetylglucosamine 2-epimerase
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DOI：
10.1021/ja021309g
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发表时间：
2003-03-05
影响因子：
15
通讯作者：
Tanner, ME
中科院分区：
文献类型：
--
作者：
Chou, WK;Hinderlich, S;Tanner, ME
双功能酶——UDP - N - 乙酰葡糖胺2 - 差向异构酶/ManNAc激酶，催化哺乳动物中唾液酸生物合成的前两步反应。差向异构酶结构域将UDP - GlcNAc转化为ManNAc和UDP。本文证明α - ManNAc是首先形成的异头物，因此该反应在C - 1位的构型净保留。在氘代缓冲液中的研究表明，在催化过程中，溶剂衍生的氘定量地掺入产物的C - 2位，但不掺入剩余的底物池中。这表明立体化学的反转最终是由C - 2位质子的去除和替换引起的，并且与双碱机制一致。用O - 18标记的UDP - GlcNAc进行的研究表明，糖基磷酸酯键的异头氧随UDP产物离去，因此净水解反应涉及C - O键的断裂。将假定的中间体2 - 乙酰氨基葡烯糖与该酶一起孵育，会发生缓慢的水合反应生成产物ManNAc。额外的动力学同位素效应和位置同位素交换（PIX）实验探讨了反应的决速步骤的性质，并表明C - H键的断裂不是限速步骤。总体而言，这些结果支持一种反应机制，即涉及UDP的反式消除生成2 - 乙酰氨基葡烯糖，随后是水的顺式加成。
The bifunctional enzyme, UDP-N-acetylglucosamine 2-epimerase/ManNAc kinase, catalyzes the first two steps in the biosynthesis of the sialic acids in mammals. The epimerase domain converts UDP-GlcNAc into ManNAc and UDP. This paper demonstrates that a-ManNAc is the first formed anomer and therefore the reaction proceeds with a net retention of configuration at C-1. Studies in deuterated buffer show that solvent-derived deuterium is quantitatively incorporated into the C-2 position of the product during catalysis, but it is not incorporated into the remaining pool of substrate. This indicates that the inversion of stereochemistry is ultimately brought about by the removal and replacement of a proton at C-2 and is consistent with a two-base mechanism. Studies with O-18-labeled UDP-GlcNAc show that the anomeric oxygen of the glycosyl phosphate bond departs with the UDP product and therefore the net hydrolysis reaction involves C-O bond cleavage. Incubation of the putative intermediate, 2-acetamidoglucal, with the enzyme resulted in a slow hydration reaction to give the product, ManNAc. Additional kinetic isotope effect and positional isotope exchange (PIX) experiments address the nature of the rate-determining step of the reaction and show that C-H bond cleavage is not rate limiting. Overall, these results support a reaction mechanism involving an anti-elimination of UDP to give 2-acetamidoglucal, followed by a syn-addition of water.