Ricin Mechanism, Transition State and Inhibitor Design

描述(由申请人提供)：动力学同位素效应应用的最新进展使理解RNA加工酶复杂相互作用的过渡态成为可能。蓖麻毒素-AB是一种异二聚体植物毒素，广泛存在于蓖麻中。B亚基是一种半乳糖特异的凝集素，它提供细胞进入复合体，然后释放A链，A链是一种腺嘌呤N-核糖水解酶，专用于28S rRNA上的单个位点。单个蓖麻毒素分子对哺乳动物细胞是致命的，几个微克对人类是致命的，使其成为最强大的细胞毒素之一。腺嘌呤底物位于RNA的发夹茎环区，终止于Gaga Tetraloop。蓖麻毒素A链的反应机理是形成一个完全解离的、具有密切相关过渡态的瞬时核氧卡宾阳离子。这一知识已被用于生产第一代蓖麻毒素A链的过渡态类似物抑制剂，并且是已知的最有效的毒素催化位点抑制剂。第二代蓖麻毒素a链的过渡态类似物将根据过渡态结构的知识进行设计。研究催化的化学机制将使用底物专一性和定点突变研究。对蓖麻毒素A链与RNA底物、过渡态和产物类似物的结构分析旨在提供关于反应配位运动和单个氨基酸在稳定过渡态复合体中的作用的信息。有效的过渡态抑制剂和用于检测蓖麻毒素A链催化活性的显色底物是本研究的两个预期产品。这些试剂可能在免疫化疗和毒素检测中用作救援剂。对蓖麻毒素A链的研究旨在为识别和共价修饰RNA的酶的反应机理提供更完整的知识。与这一目标一致，将启动对RNA位点特异性腺苷脱氨酶ADAR过渡态结构的研究。在信使核糖核酸中的腺苷酸处的脱氨基产生一个被翻译为G的肌苷位点，从而引起A&gt；G密码子的改变。这些酶的过渡态抑制剂有望在改变蛋白质表达和病毒感染性方面找到用途。

DESCRIPTION (provided by applicant): Recent advances in the application of kinetic isotope effects makes it possible to understand transition states for the complex interactions of RNA processing enzymes. Ricin-AB is a heterodimeric plant toxin, abundant in the castor bean. Subunit B is a galactose-specific lectin that provides cell entry to the complex followed by release of the A-chain, an adenine N-ribohydrolase specific for a single site on 28S rRNA. A single molecule of ricin is lethal for a mammalian cell, and a few ug are lethal for a human, making it among the most powerful cytotoxins. The adenine substrate resides in a hairpin stem-loop region of RNA, terminating in a GAGA tetraloop. The reaction mechanism for ricin A-chain forms a fully dissociated and transient ribooxacarbenium ion with closely related transition states. This knowledge has been used to produce the first generation of transition state analogue inhibitors for ricin A-chain, and is the most powerful catalytic site inhibitors known for the toxin. Second-generation transition state analogues for ricin a-chain will be designed from knowledge of the transition state structure. Investigation of the chemical mechanism of catalysis will use substrate specificity and site-directed mutagenesis studies. Structural analysis of ricin A-chain in complex with substrate, transition state and product analogues of RNA is intended to provide information on reaction coordinate motion and the role of individual amino acids in stabilizing the transition state complex. Powerful transition state inhibitors and chromogenic substrates for detecting ricin A-chain catalytic activity are two anticipated products of the research. These agents may be of use as rescue agents in immunochemotherapy and in detection of the toxin. The studies of ricin A-chain are intended to provide more complete knowledge of the reaction mechanisms for enzymes that recognize and covalently modify RNA. Consistent with this goal, studies will be initiated toward the transition state structure of an RNA site-specific adenylate deaminase, ADAR. Deamination at an adenylate site in mRNA produces an inosine site that is translated as a G, therefore causing A > G codon changes. Transition state inhibitors for these enzymes are anticipated to find use in altering protein expression and viral infectivity.