Effect of a chemical modification on the hydrated adenosine intermediate produced by adenosine deaminase and a model reaction for a potential mechanism of action of 5-aminoimidazole ribonucleotide carboxylase.
Effect of a chemical modification on the hydrated adenosine intermediate produced by adenosine deaminase and a model reaction for a potential mechanism of action of 5-aminoimidazole ribonucleotide carboxylase.
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化学修饰对腺苷脱氨酶产生的水合腺苷中间体的影响以及 5-氨基咪唑核糖核苷酸羧化酶潜在作用机制的模型反应。
DOI：
10.1021/jm970301s
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Robinson,PD
中科院分区：
文献类型：
--
作者：
Groziak,MP;Huan,ZW;Ding,H;Meng,Z;Stevens,WC;Robinson,PD
以腺苷脱氨酶（ADA，EC 3.5.4.4）产生的水合腺苷中间体（6R）-6-氨基-1,6-二氢-6-羟基-9-（β-d-呋喃核糖基）嘌呤（2）为起点，通过去除−C6(OH)(NH2)− 由酶催化水解的早期事件产生的 2 分子片段。 FAIRs 是直接从 5-氨基-1-(β-d-呋喃核糖基)咪唑-4-羧酸 (CAIR) 的钠盐合成的，反应顺序涉及串联 N-甲酰化/脱羧，这可能与大肠杆菌 E 催化的 N5-CAIR 异构化为 CAIR 具有机械联系。阐明了 FAIR 的物理和光谱性质，确定了其 X 射线晶体和 NMR 溶液结构，并研究了其与 ADA 的相互作用。结晶FAIR仅作为Z-甲酰胺旋转异构体存在，并且表现出许多已知有助于Ado与ADA缔合的相同的分子内氢键合事件。然而，在水和各种有机溶剂中，FAIR 以 NMR 不同、缓慢相互转化的 Z 和 E 异构体的形式存在。这种截短的酶促四面体中间类似物被确定为 ADA 的竞争性抑制剂，其表观 Ki 结合常数为 40 μM，该值非常接近天然底物的 Km 值 (33 μM)。然而，选择用于 ADA 结合的实际物种很可能是 Z-FAIR 的次要羟亚氨基质子型形式，其真实 Ki 值要低得多。由于 FAIR 的结构特征似乎非常适合支持其用作构建 ADA 和 AIR 羧化酶活性位点探针的模板，因此合成了适合轻松糖苷精制的各种碳水化合物保护版本的 FAIR。对其中一些的 N3-烷基化、N3-硼烷络合和 C4-碘化进行了研究，以评估可能有助于阐明 AIR 羧化酶机制的物理化学性质。对这些特性的调查与模型 CAIRs → FAIRs 合成转化的合理机制一起被解释为支持纯 E 催化的 N5-CAIR → CAIR 生物合成机制，该机制涉及咪唑 C4 原子的羧化 sp3-重新杂化，而不是具有偶极稳定的 C4 sp2 碳阴离子中间体。
Using the hydrated adenosine intermediate (6R)-6-amino-1,6-dihydro-6-hydroxy-9-(β-d-ribofuranosyl)purine (2) produced by adenosine deaminase (ADA, EC 3.5.4.4) as a starting point, the active site probe and inhibitor platform 5-(formylamino)imidazole riboside (FAIRs,4) was designed by removal of the −C6(OH)(NH2)− molecular fragment of2generated by the early events of the enzyme-catalyzed hydrolysis. FAIRs was synthesized directly from the sodium salt of 5-amino-1-(β-d-ribofuranosyl)imidazole-4-carboxylic acid (CAIR) along a reaction sequence involving a tandemN-formylation/decarboxylation that may have a mechanistic connection to theEscherichia colipurE-catalyzed constitutional isomerization ofN5-CAIR to CAIR. The physical and spectral properties of FAIRs were elucidated, its X-ray crystal and NMR solution structures were determined, and its interaction with ADA was investigated. Crystalline FAIRs exists solely as theZ-formamide rotamer and exhibits many of the same intramolecular hydrogen bonding events known to contribute to the association of Ado to ADA. In water and various organic solvents, however, FAIRs exists as NMR-distinct, slowly interconvertingZandErotamers. This truncated enzymatic tetrahedral intermediate analog was determined to be a competitive inhibitor of ADA with anapparentKibinding constant of 40 μM, a value quite close to that (33 μM) of the natural substrate'sKm. The actual species selected for binding by ADA, though, is likely the minor hydroxyimino prototropic form ofZ-FAIRs possessing a far lower trueKivalue. As the structural features of FAIRs appear well-suited to support its use as a template for constructing active site probes of both ADA and AIR carboxylases, a variety of carbohydrate-protected versions of FAIRs suitable for facile aglycon elaborations were synthesized. TheN3-alkylation,N3-borane complexation, andC4-iodination of some of these were investigated in order to assess physicochemical properties that may assist in the elucidation of mechanisms for the AIR carboxylases. The survey of these properties taken together with a reasonable mechanism for the model CAIRs → FAIRs synthetic transformation is interpreted to support a mechanism for thepurE-catalyzedN5-CAIR → CAIR biosynthetic one that involves a carboxylative sp3-rehybridization of the imidazole C4 atom rather than one possessing a dipole-stabilized C4 sp2carbanionic intermediate.