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得理解RNA加工酶复杂相互作用的过渡态成为可能。蓖麻毒素-AB是一种异源二聚体植物毒素，在蓖麻子中含量丰富。亚基B是一种半乳糖特异性凝集素，可使细胞进入复合物，然后释放A链，A链是一种对28 S rRNA上的单个位点具有特异性的腺嘌呤N-核糖水解酶。一个蓖麻毒素分子对哺乳动物细胞是致命的，几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四环。蓖麻毒蛋白A链的反应机理是形成一个完全解离的、瞬时的、具有密切相关过渡态的核糖氧碳烯鎓离子。这一知识已被用于生产蓖麻毒素A链的第一代过渡态类似物抑制剂，并且是已知的毒素的最强大的催化位点抑制剂。第二代过渡态类似物蓖麻毒素a-链将设计从知识的过渡态结构。催化的化学机制的调查将使用底物特异性和定点诱变研究。蓖麻毒素A链与RNA的底物、过渡态和产物类似物的复合物的结构分析旨在提供关于反应坐标运动和单个氨基酸在稳定过渡态复合物中的作用的信息。强有力的过渡态抑制剂和检测蓖麻毒素A链催化活性的显色底物是该研究的两个预期产品。这些试剂可用作免疫化疗和毒素检测中的救援剂。蓖麻毒素A链的研究旨在为识别和共价修饰RNA的酶的反应机制提供更完整的知识。与这一目标相一致，研究将开始对过渡态结构的RNA位点特异性腺苷酸脱氨酶，阿达尔。mRNA中腺苷酸位点的脱氨基作用产生一个被翻译为G的肌苷位点，因此导致A> G密码子改变。预期这些酶的过渡态抑制剂可用于改变蛋白质表达和病毒感染性。

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.