Isozyme-specific transition state inhibitors for the trypanosomal nucleoside hydrolases.
Isozyme-specific transition state inhibitors for the trypanosomal nucleoside hydrolases.
复制标题
锥虫核苷水解酶的同工酶特异性过渡态抑制剂。
DOI：
10.1021/bi962319v
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Schramm,VL
中科院分区：
文献类型：
--
作者：
Parkin,DW;Limberg,G;Tyler,PC;Furneaux,RH;Chen,XY;Schramm,VL
原生动物寄生虫缺乏从头开始的嘌呤生物合成，需要从宿主那里回收嘌呤。核苷水解酶参与核苷回收，在哺乳动物中未发现，这使其成为抑制剂设计的原生动物特异性靶点。几种具有不同底物特异性的原生动物核苷水解酶同工酶已得到表征。新型取代亚氨基核糖醇已被合成，类似于来自 Crithidiafasciculata 的非特异性肌苷-尿苷核苷水解酶（IU-核苷水解酶）的过渡态结构。这些抑制剂已针对该酶和来自布氏锥虫的嘌呤特异性核苷水解酶（IAG-核苷水解酶）进行了表征。为IU-核苷水解酶提供纳摩尔抑制常数的抑制剂对IAG-酶表现出微摩尔抑制常数。例如，对溴苯基亚氨基核糖醇抑制 IU 酶和 IAG 酶，解离常数分别为 28 nM 和 190 μM。底物特异性、过渡态抑制剂的作用以及动力学常数的 pH 依赖性表明 IU- 和 IAG- 同工酶的催化机制和过渡态结构根本不同。这一发现是引人注目的，因为这些同工酶在催化位点上具有显着的同源性，并且都使用肌苷作为优选底物。过渡态类似物的特异性表明，合理设计的过渡态抑制剂具有同工酶特异性，(Km/KiIU-核苷水解酶)/(Km/KiIAG-核苷水解酶)值高达39 000。差异抑制的机制基于这些酶的相对离去基团活化和核糖基-氧碳鎓形成能力。除了提供同工酶特异性抑制剂之外，本文描述的新型分子对于 N-核糖水解酶的过渡态性质具有诊断价值。
Protozoan parasites lack de novo purine biosynthesis and require purine salvage from the host. Nucleoside hydrolases are involved in nucleoside salvage and are not found in mammals, making them protozoan-specific targets for inhibitor design. Several protozoan nucleoside hydrolase isozymes with distinct substrate specificities have been characterized. Novel substituted iminoribitols have been synthesized to resemble the transition state structure of the nonspecific inosine−uridine nucleoside hydrolase fromCrithidiafasciculata(IU-nucleoside hydrolase). These inhibitors have been characterized for this enzyme and for a purine-specific nucleoside hydrolase (IAG-nucleoside hydrolase) fromTrypanosoma brucei brucei. Inhibitors which provide nanomolar inhibition constants for IU-nucleoside hydrolase exhibit micromolar inhibition constants for the IAG-enzyme. For example,p-bromophenyliminoribitol inhibits the IU- and IAG-enzymes with dissociation constants of 28 nM and 190 μM, respectively. Substrate specificity, the action of transition state inhibitors and the pH-dependence of the kinetic constants establish that the catalytic mechanisms and transition state structures are fundamentally different for the IU- and IAG-isozymes. The finding is remarkable since these isozymes share significant homology at the catalytic sites and both use inosine as a preferred substrate. The specificity of the transition state analogues indicates that logically-designed transition state inhibitors are isozyme-specific, with (Km/KiIU-nucleoside hydrolase)/(Km/KiIAG-nucleoside hydrolase) values up to 39 000. The mechanism of the differential inhibition is based on the relative leaving group activation and ribosyl−oxocarbenium-forming abilities of these enzymes. In addition to providing isozyme-specific inhibitors, the novel molecules described here have diagnostic value for the nature of the transition states forN-ribohydrolase enzymes.