NEW CHEMICAL APPROACHES TO ADP-RIBOSYL TRANSFER

ADP-核糖基转移酶和相关的NAD糖水解酶是一系列 催化二磷酸腺苷核糖转移的酶的种类 NAD的一部分可与多种亲核受体或水结合。这个 这种翻译后修饰的生物学意义 尚不清楚，尽管已经有几个重要的监管角色 建议。在此提出了一系列的设计方案。 被预测为“自杀式”酶失活剂的化合物 (即作为基于机理的不可逆酶抑制剂)用于NAD 糖水解酶和相关的ADP-核糖基转移酶。它的作用机制 抑制作用利用了已有文献记载的吡啶碱基交换活性 NAD糖水解酶。首先将努力获得 必要的化合物，并确定它们是否真的是“自杀” 已知的进行吡啶碱基交换的酶的灭活剂 反应。接下来，将尝试扩展这种类型的抑制 与各种ADP-核糖基转移酶结合。这样的抑制剂将在 标记和鉴定敏感酶中的活性部位残基。一次 目标酶已经确定，这些抑制剂将用于 探索培养细胞中靶标的生理功能(S)。 另一种抑制剂设计策略是提出的，这一策略涉及 生产一种“不可切割”的NAD类似物，它被预测为一种 ADP-核糖基转移酶的非共价抑制剂。这样的化合物将会 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的多(ADP-核糖)合成酶。其他 提出了对该酶进行的机理实验。这些字符以 建立了一种灵敏的连续测定方法，并随后进行了仔细的 旨在检测反应中任何中间体的动力学研究。

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.