A transition state in pieces: major contributions of entropic effects to ligand binding by adenosine deaminase.
A transition state in pieces: major contributions of entropic effects to ligand binding by adenosine deaminase.
复制标题
碎片的过渡状态：熵效应对腺苷脱氨酶配体结合的主要贡献。
DOI：
10.1021/bi00147a021
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Wolfenden,R
中科院分区：
文献类型：
--
作者：
Kati,WM;Acheson,SA;Wolfenden,R
修订稿于 1992 年 4 月 9 日收到摘要：星云碱在腺苷脱氨酶的活性位点发生水合作用，该反应类似于用水从腺苷中置换氨的部分反应，生成抑制复合物，该复合物捕获了理想过渡态类似物预期的大部分结合亲和力。比较了与星云碱 1, 6-水合物及其稳定类似物 2'-脱氧福霉素相关的几种化合物的酶亲和力，以确定强结合的结构起源。通过去除取代基核糖，使稳定过渡态类似物抑制剂 2'-脱氧考福霉素的结合变得不太有利，通过去除取代基核糖，使结合变得不太有利，通过倒置二氮杂环的 8-羟基取代基，使结合变得不太有利，并且通过去除二氮杂环的 4-6 原子，使结合变得不太有利，使结合减少 10.0 kcal/mol。通过去除6-羟基基团，使不稳定的过渡态类似物星云水合物的结合变得不那么有利至少9.9 kcal/mol，并且通过去除嘧啶环的1-3原子使结合变得不那么有利，至少10.2 kcal/mol。在每种情况下，酶对“缺失的片段”仅表现出适度的亲和力（Ki> 10™ 2 M），这表明在单个分子中掺入 2 个结合决定簇允许额外的 7-12 kcal/mol 内在结合能表现为观察到的结合能。这些结果与早期的迹象一致，即腺苷脱氨酶可以利用两种底物结合的内在自由能10.5 kcal/mol将它们放置在适合于活性位点反应的位置，克服对嘌呤核糖核苷的1, 6-水合平衡不利的-35 eu熵变，并降低腺苷脱氨中达到过渡态的平衡常数。因此，腺苷脱氨酶通过在其活性位点将非酶促双分子水合反应转化为单分子反应，可以将其催化能力提高高达 8 个数量级。几种新的 6-取代 1, 6-二氢嘌呤核糖核苷，通过甲酸盐光加成和低温添加有机锂试剂到嘌呤核糖核苷衍生物中制备，对腺苷脱氨酶表现出 9-1400 µ 的 Ki 值，这与活性位点对底物中庞大的离去基团的相当大的耐受性一致。 6-羧基-1, 6-二氢嘌呤核糖核苷的一种非对映异构体的抑制被发现具有时间依赖性，从弱结合发展到更强结合的复合物。过渡态类似物抑制剂为研究酶机制提供了有用的结构工具，因为对其抑制复合物的检查可以提供有关可能参与催化的结合相互作用的证据。就腺苷脱氨酶而言，竞争性抑制剂星云碱（nebularine）在 C-6 处缺乏取代基，不会以游离溶液中丰富的形式结合（1，方案 I），而是以 1, 6-加成化合物的形式结合（Kurz & Frieden, 1987）。通过将其 UV 和 NMR 光谱与相对稳定的类似物（其中其 N-1 氢原子已被甲基取代）进行比较，该结合加成化合物已被鉴定为 neb-ularine 1, 6-水合物（2，方案），类似于括号中的水合反应中间体（Jones 等人，1989），酶-抑制剂复合物单晶的 X 射线衍射证实了这一分配（Wilson 等人， 1991）。星云碱 1、6-水合物、3 X 10™ 13 M（Jones 等人，1989）的 K 值远低于产品肌苷 1 X 10-4 M 的 K 值，并且与最大解离常数相差不远（约 10-16）
Revised Manuscript Received April 9, 1992 abstract: Nebularine undergoes hydration at theactive site of adenosine deaminase, in a reaction analogous to a partial reaction in the displacement of ammonia from adenosine by water, to generate an inhibitory complex that captures much of the binding affinity expected of an ideal transition-state analogue. Enzyme affinities of several compounds related to nebularine 1, 6-hydrate, and to its stable analog 2'-deoxyco-formycin, were compared in an effort to identify the structural origins of strong binding. Binding of the stable transition-state analog inhibitor 2'-deoxycoformycin was rendered 9.8 kcal/mol less favorable by removal of substituent ribose, 9.7 kcal/mol less favorable by inversion of the 8-hydroxyl substituent of the diazepine ring, and 10.0 kcal/mol less favorable by removal of atoms 4-6 of the diazepine ring. Binding of the unstable transition-state analog nebularine hydrate was rendered at least 9.9 kcal/mol less favorable by removal of the 6-hydroxyl group and 10.2 kcal/mol less favorable by removal of atoms 1-3 of the pyrimidine ring. In each case, the enzyme exhibited only modest affinity (Ki> 10™ 2 M) for the “missing piece”, indicatingthat incorporation of 2 binding determinants within a single molecule permits an additional 7-12 kcal/molof intrinsic binding energy to be manifested as observed binding energy. These results are consistent with earlier indications that adenosine deaminase may use 10.5 kcal/mol of the intrinsic free energy of binding of the two substrates to place them in positions appropriate for reaction at the activesite, overcoming the unfavorable entropy change of-35 eu for the equilibrium of 1, 6-hydration of purine ri-bonucleoside and reducing the equilibrium constant for attainment of the transition state in deamination of adenosine. Thus, adenosine deaminase may achieve up to 8 orders of magnitude of its catalytic power by converting the nonenzymatic, bimolecular, hydration reaction to a monomolecular reaction at its active site. Several new 6-substituted 1, 6-dihydropurine ribonucleosides, prepared by photoaddition of formate and by low-temperature addition of organolithium reagents to a derivative of purine ribonucleoside, exhibited Ki values of 9-1400 µ against adenosine deaminase, in accord with the active site’s considerable tolerance of bulky leaving groups in substrates. Inhibition by one diastereomer of 6-carboxy-1, 6-dihydropurine ri-bonucleoside was found to be time-dependent, progressing from a weakly bound to a more strongly bound complex.Transition-state analog inhibitors offer a useful structural tool for studying enzyme mechanisms, because examination of their inhibitory complexes can supply evidence concerning binding interactions that are likely to be involved in catalysis. In the case of adenosine deaminase, the competitive inhibitor nebularine, lacking a substituent at C-6, is not bound in the form that is abundant in free solution (1, Scheme I), but rather as a 1, 6-addition compound (Kurz & Frieden, 1987). This bound addition compound has been identified as neb-ularine 1, 6-hydrate (2, Scheme), resembling the hydrated reaction intermediate in brackets, by comparison of its UV and NMR spectra with those of the relatively stable analog in which its N-1 hydrogen atom has been replaced by a meth-yl group (Jones et al., 1989), and X-ray diffraction from single crystals of the enzyme-inhibitor complex confirms this assignment (Wilson et al., 1991). The K\value of nebularine 1, 6-hydrate, 3 X 10™ 13 M (Jones et al., 1989), is much lower than thatof the product inosine, 1 X 10-4 M, and is not far removed from the maximal dissociation constant (ca. 10-16