Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine.
Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine.
复制标题
9-β-D-阿拉伯呋喃糖腺嘌呤的生物合成：腺苷 C-2 处的氢交换和 C-3 处的氧交换。
DOI：
10.1016/0003-9861(89)90039-8
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发表时间：
1989
影响因子：
3.9
通讯作者：
Hebbler,AK
中科院分区：
文献类型：
--
作者：
Suhadolnik,RJ;Pornbanlualap,S;Wu,JM;Baker,DC;Hebbler,AK
本文报道了利用链霉菌（Streptomyces arcticus）在体内研究和部分纯化的酶催化腺苷转化为9-β-d-阿拉伯呋喃糖基腺嘌呤（ara-A）的新发现。首先，在[2′-18 O]-和[U-14 C]-腺苷的双标记体内实验中，分离的ara-A的18 O：14 C比例没有明显变化，表明腺苷的C-2′羟基立体特异性转化为ara-A，18 O保留在C-2′。在[3′-18 O]-和[U-14 C]-腺苷的实验中，分离出[U-14 C]ara-A;然而，C-3′的18 O低于检测值。从两个双标记实验的RNA中分离的腺苷具有基本上相同的18 O：14 C比率。第二，已经从S.催化腺苷转化为ara-A，但不催化AMP、ADP、ATP、肌苷、鸟苷、邻-核糖转化为ara-A。在用[U-14 C]腺苷进行的单标记酶催化实验中，有9.9%转化为[U-14 C]ara-A;用[2′-3 H]-腺苷，有8.9%的C-2′氚从[2′-3 H]腺苷中释放出来，以3 H2O形式回收。第三，通过将该酶与3 H2O和腺苷一起孵育，证实了3 H以3 H2O形式从[2′-3H]腺苷中释放。90%的氚掺入到分离的ara-A的d-阿拉伯糖中，在C-2中，8%在C-3中。酶催化的腺苷转化为ara-A的过程不需要添加辅助因子，表现出饱和动力学，最适pH为6.8，aKm为8 × 10− 4 M，重金属阳离子会抑制。该酶还催化核苷抗生素结核菌素的C-2′羟基立体特异性转化，形成7-β-d-阿拉伯呋喃糖基-4-氨基吡咯并[2，3-d]嘧啶。核苷类抗生素桑吉瓦霉素（sangivamycin）的C-5位氢原子被甲酰胺基团取代，它不是底物。在单、双标记实验和体外酶催化实验的基础上，提出了3′-酮核苷中间体和自由基阳离子两种机理来解释观察到的数据。
The data presented here describe new findings related to the bioconversion of adenosine to 9-β-d-arabinofuranosyladenine (ara-A) byStreptomyces antibioticusbyin vivoinvestigations and with a partially purified enzyme. First, in double labelin vivoexperiments with [2′-18O]- and [U-14C]adenosine, the18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2′ hydroxyl of adenosine to ara-Awith retentionof the18O at C-2′. In experiments with [3′-18O]- and [U-14C]-adenosine, [U-14C]ara-A was isolated; however, the18O at C-3′ is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of18O:14C. Second, an enzyme has been isolated and partially purified from extracts ofS. antibioticusthat catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, ord-ribose, to ara-A. In a single label enzyme-catalyzed experiment with [U-14C]adenosine, there was a 9.9% conversion to [U-14C]ara-A; with [2′-3H]-adenosine, there was a 8.9% release of the C-2′ tritium from [2′-3H]adenosine which was recovered as3H2O. Third, the release of3H as3H2O from [2′-3H]adenosine was confirmed by incubations of the enzyme with3H2O and adenosine. Ninety percent of the tritium incorporated into thed-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, aKmof 8 × 10−4M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2′ hydroxyl of the nucleoside antibiotic, tubercidin to form 7-β-d-arabinofuranosyl-4-aminopyrrolo[2,3-d]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experimentsin vivoand thein vitroenzyme-catalyzed experiments, two mechanisms involving either a 3′-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.