Binding modes for substrate and a proposed transition-state analogue of protozoan nucleoside hydrolase.
Binding modes for substrate and a proposed transition-state analogue of protozoan nucleoside hydrolase.
复制标题
底物的结合模式和原生动物核苷水解酶的拟议过渡态类似物。
DOI：
10.1021/bi00042a030
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Schramm,VL
中科院分区：
文献类型：
--
作者：
Parkin,DW;Schramm,VL
1995年8月24日收到的修订稿件？摘要：丛枝克里迪亚的肌苷-尿苷核苷水解酶(IU-核苷水解酶)的过渡态结构的特征是核糖中的氧碳正离子特征以及与离开的次黄嘌呤和初始亲水亲核分子的弱键[Horenstein，B.A.，Parkin，D.W.，Estupiñán，B.，&Schramm，VL(1991年)生化30,10788-10795]，旨在类似于过渡态的抑制物是缓慢起效的，紧密结合的抑制剂，观察到的KjK值高达2×105[Schramm，VL，Horenstein，BH，Kline，P.C.(1994)J.Biol.化学。269,18259-18262]，虽然缓慢起效的紧密结合与过渡态稳定是一致的，但通过比较与底物相互作用以提供结合和催化的基团与那些与假定的过渡态抑制物相互作用的基团，可以获得更直接的证据。肌苷与IU-核苷水解酶结合的Km值在5.6-10.5范围内与pH无关。Vmax和VmiJKm对pH的依赖性导致pH最适接近8.0。PK为9.1的单一基团必须质子化才有催化活性，而pK为7.1的第二个基团的质子化会导致活性丧失。1-(S)-苯基-1，4-二脱氧-1，4-亚氨基-D-核糖醇(苯亚胺-D-核糖醇)与30 nm的平衡Kt‘结合，被认为是一种过渡态的抑制剂。苯亚胺核糖醇竞争抑制的pH依赖关系类似于Vmax曲线，抑制剂结合所需的单个基团的质子化pK为7.5，而随后的基团的质子化pK为6.6，导致结合的丧失。有人提出，质子化抑制剂(pK 6.5)的正电荷是结合为过渡态抑制剂的识别特征。然而，pH分析表明，中性抑制剂是结合该酶活性形式的首选物种。苯亚胺核糖醇结合的缓慢起效阶段在低pH值下消失，这表明结合复合体的依赖于时间的质子化可能是抑制作用缓慢起效阶段的原因。来自支链孢子虫的肌苷-尿苷偏好核苷水解酶(IU-核苷水解酶)水解常见的嘌呤和嘧啶核苷的N-糖苷键(Parkin等人，1991年)。动力学同位素效应研究已经证实，肌苷的酶稳定过渡状态不同于酸催化的溶剂分解过程中出现的二质子化的嘌呤(Horenstein等人，1991；Garrett&Mehta，1972；Cherian等人，1990)。基于肌苷和肌苷在过渡态的几何和静电势面(Horenstein&Schramm，1993a)，一系列包含过渡态的各种特征的抑制剂被制备(Horenstein&Schramm，1993b；Horenstein等人，1993；Boutellier等人，1994)。这些抑制剂的一个共同特征是能够被质子化以形成核氧碳正离子模拟物，并与与
Revised Manuscript Received August 24, 1995® abstract: The transition-state structure for inosine—uridine nucleoside hydrolase (IU-nucleoside hydrolase) from Crithidia fasciculata is characterized by oxycarbonium character in the ribosyl and weak bonds to the departing hypoxanthine and incipient water nucleophile [Horenstein, B. A., Parkin, D. W., Estupiñán, B., & Schramm, VL (1991) Biochemistry 30, 10788—10795], Inhibitors designed to resemble the transition state are slow-onset, tight-binding inhibitorswith observed KJK\values up to 2 x 105 [Schramm, VL, Horenstein, BH, & Kline, P. C.(1994) J. Biol. Chem. 269, 18259—18262], Although slow-onset, tight binding is consistent with transition-state stabilization, more direct evidence can be obtained by comparing the groups which interact with the substrate to provide binding and catalysis with those which interact with the putative transition-state inhibitor. The Km value for inosine binding to IU-nucleoside hydrolase is independent of pH over the range 5.6—10.5. Dependencies of Vmax and VmiJKm on pH result in pH optima near 8.0. A single group with pK of 9.1 must be protonated for catalytic activity, and protonation of a second group with a pK of 7.1 results in loss of activity. 1-(S)-Phenyl-1, 4-dideoxy-1, 4-imino-D-ribitol (phenyliminoribitol) binds with an equilibrium Kt¡ of 30 nM and has been proposed to be a transition-state inhibitor. The pH dependence for the competitive inhibition by phenyliminoribitol resembles the Vmax profile with the protonation of a single group, pK 7.5, required for inhibitor binding and the protonation of a subsequent group, pK 6.6, causing loss of binding. It has been proposed that the positive charge of protonated inhibitor (pK 6.5) is a recognition feature for binding as a transition-state inhibitor. However, the pH analysis indicates thatthe neutral inhibitor is the preferred species for binding the active form of the enzyme. The slow-onset phase of phenyliminoribitol binding disappears at low pH, suggesting that a time-dependent protonation of the bound complex could be responsible for the slow-onset phase of inhibition.The inosine—uridine preferring nucleoside hydrolase (IU-nucleoside hydrolase) from Crithidia fasciculata hydrolyzes the N-glycosidic bonds of the commonly occurring purine and pyrimidine nucleosides (Parkin et al., 1991). Kinetic isotope effect studies have established that the enzymestabilized transition state for inosine is distinct from the diprotonated purine which occurs during acid-catalyzed solvolysis (Horenstein et al., 1991; Garrett & Mehta, 1972; Cherian et al., 1990). Based on the geometry and electro-static potential surfaces of inosine and inosine at the transition state (Horenstein & Schramm, 1993a), a family of inhibitors were prepared which contain various features of the transition state (Horenstein & Schramm, 1993b; Horenstein et al., 1993; Boutellier et al., 1994). A common feature of these inhibitors is the ability to be protonated to form ribooxycarbonium mimics and tight binding to the enzymerelative to the