NEW CHEMICAL APPROACHES TO ADP-RIBOSYL TRANSFER

ADP-核糖基转移的新化学方法

• 批准号: 3281962
• 负责人: JAMES T SLAMA
• 金额: $ 6.75万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 1986-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3281962
• 关键词: NAD nucleosidase; NAD(H) analog; chemical synthesis; enzyme substrate analog; ligase; molecular site; nicotinamide adenine dinucleotide; nucleotidyltransferase; tissue /cell culture; ultraviolet spectrometry

ADP-ribosyl transferases and the related NAD glycohydrolases are a series of enzymes which catalyze the transfer of the adenosine diphosphate ribose portion of NAD to a variety of nucleophilic acceptors or to water. The biological significance of this variety of posttranslational modifications is not yet clear, although several important regulatory roles have been suggested. A proposal is advanced herein for the design of a series of compounds which are predicted to function as "suicide" enzyme inactivators (i.e., as mechanism based irreversible enzyme inhibitors) for NAD glycohydrolases and related ADP-ribosyl transferases. The mechanism of inhibition exploits the well documented pyridine base exchange activity of the NAD glycohydrolases. An effort first will be made to obtain the requisite compounds and to determine whether they are indeed "suicide" inactivators for enzymes known to carry out the pyridine base exchange reaction. Next, an attempt will be made to extend this type of inhibition to various ADP-ribosyl transferases. Such inhibitors will be useful in labeling and identifying active site residues in susceptible enzymes. Once the target enzymes have been identified, the inhibitors will be used to probe the physiological function(s) of the targets in cultured cells. Another strategy for inhibitor design is advanced, this one involving the production of a "non-cleavable" NAD analog which is predicted to be a non-covalent inhibitor of ADP-ribosyl transferases. Such a compound would be valuable for in vitro studies on ADP-ribosyl transferases, particularly the poly(ADP-ribose) synthetase isolated from mammalian nuclei. Other mechanistic experiments on this enzyme are proposed. These begin with the development of a sensitive continuous assay and a subsequent careful kinetic study designed to detect any intermediates in the reaction.

ADP-核糖基转移酶和相关的NAD糖水解酶是一系列 催化腺苷二磷酸核糖转移的酶 一部分NAD与各种亲核受体或水反应。 的 这些翻译后修饰的生物学意义 目前还不清楚，尽管已经有几个重要的监管角色， 建议。 本文提出了一种设计一系列 被预测作为“自杀”酶灭活剂的化合物 (i.e.,作为基于机制的不可逆酶抑制剂） 糖水解酶和相关的ADP-核糖基转移酶。 的机理 抑制利用了充分记录的吡啶碱交换活性， NAD糖水解酶。 首先将努力获得 必要的化合物，并确定它们是否确实是“自杀” 用于已知进行吡啶碱交换的酶的灭活剂 反应 接下来，将尝试扩展这种类型的抑制 各种ADP-核糖基转移酶。 这样的抑制剂将可用于 标记和鉴定敏感酶中的活性位点残基。 一旦 目标酶已经确定，抑制剂将用于 探测培养细胞中靶标的生理功能。 抑制剂设计的另一种策略是先进的，这一个涉及 产生“不可裂解的”NAD类似物，其被预测为 ADP-核糖基转移酶的非共价抑制剂。 这种化合物将 对ADP-核糖基转移酶的体外研究有价值，特别是 从哺乳动物细胞核中分离出的聚（ADP-核糖）合成酶。 其他 提出了对该酶的机理实验。 这些开始与 开发一种灵敏的连续检测方法， 动力学研究旨在检测反应中的任何中间体。