NEW CHEMICAL APPROACHES TO ADP-RIBOSYL TRANSFER

ADP-核糖基转移酶是一类重要的调节酶。 该项目提出了NAD专用的设计和亲和力标签 ADP-核糖基转移酶的底物结合部位。这些信息 寻求的是催化必需氨基酸的鉴定 残基和活性部位结构的阐明。身份识别 必需氨基酸残基和活性部位结构的阐明。 必需氨基酸残基的鉴定将促进 了解这些独特催化剂的机理。这 信息将支持基因工程的发展 百日咳和相关疾病的疫苗，其中微生物ADP- 核糖基转移酶是一种外毒素成分。 亲和标记和光亲和标记将被应用于 哺乳动物NAD糖水解酶作为水解性转移酶的例子 以及具有催化活性的。霍乱毒素A亚单位百日咳 毒素和白喉毒素。这些毒素在医学上很重要，而且 作为单(ADP-核糖基)的代表具有重要意义 被认为是一种新的细胞调节因子的转移酶 机制。亲和标记和光亲和标记都不是特定于 NAD底物结合部位，一般适用于 类当前可用。所有这些酶都表现出显著的NAD 糖水解酶活性。因此，成功的标签必须是不可分割的 NAD类似物。生产亲和标签的第一种方法 而光亲和标记因此成为可能，因为我们开发了 不可切割的类似物。生产的第一种方法 因此，亲和标记和光亲和标记通过以下方式成为可能 我们开发的非切割类似物Carba-NAD。在这里a 环戊烷环取代烟酰胺的β-D-核糖肽 NAD中的核苷部分。 Carba-NAD已被证明对ADP-核糖转移具有抵抗力 并成为NAD糖水解酶的有效竞争性抑制剂和 几种单(ADP-核糖基)转移酶。一个有亲缘关系的家庭 标签和亲光性标签将通过对 Carba-NAD，并应用于上述酶的研究。一个 抑制剂设计的第二个策略将利用氨基糖作为 模拟氧碳正离子中间体的“过渡态类似物” 建议用于哺乳动物NAD糖水解酶。1，4-二氢呋喃的合成 二脱氧-4-氨基-D-呋喃核糖是可用的，并结合这一 将氨基糖转化为ADP-核糖类似物将很容易完成。这个 “过渡态类似物”将作为NAD的抑制剂进行测试-- 糖水解酶和微生物毒素。

ADP-ribosyl transferases are an important class of regulatory enzymes. This project proposes the design and affinity labels specific for the NAD substrate binding site of ADP-ribosyl transferases. The information sought is the identification of catalytically essential amino acid residues and elucidation of active site structure. Identification of essential amino acid residues and elucidation of active site structure. Identification of essential amino acid residues will advance the understanding of the mechanism of these unique catalysts. This information will support the development of genetically engineered vaccines against pertussis and related diseases where a microbial ADP- ribosyl transferase functions as an exotoxin component. Affinity and photoaffinity labels will be applied to the study of the mammalian NAD glycohydrolase as an example of a hydrolytic transferase and to the catalytically active . A subunits of cholera toxin, pertussis toxin, and diphtheria toxin. These toxins are medically important and are significant as representatives of the class of mono (ADP-ribosyl) transferases which has been implicated as a new cell regulatory mechanism. Neither affinity labels nor photoaffinity labels specific for the NAD substrate binding site and generally applicable to enzymes in the class are currently available. All such enzymes exhibit significant NAD glycohydrolase activity. Successful labels must therefore be noncleavable NAD analogues. The first approach to the production of affinity labels and photoaffinity labels is therefore made possible by our development of the non-cleavable analogues. The first approach to the production of affinity labels and photoaffinity labels is therefore made possible by our development of the non-cleavable analogue carba-NAD. Here a cyclopentane ring replaces the beta-D-ribotide of the nicotinamide riboside moiety of NAD. Carba-NAD has been demonstrated to be resistant to ADP-ribosyl transfer and to be an effective competitive inhibitor of NAD glycohydrolase and several mono (ADP-ribosyl) transferases. A family of related affinity labels and photoaffinity labels will be produced by modification of carba-NAD and applied to the study of the aforementioned enzymes. A second strategy for inhibitor design will utilize amino sugars as "transition-state analogues" to mimic the oxocarbonium ion intermediate proposed for the mammaliam NAD glycohydrolase. A synthesis of 1,4- dideoxy-4-amino-D-ribofuranose is available and incorporation of this amino sugar into an ADP-ribose analogue will readily be accomplished. The "transition-state analogue" will be tested as an inhibitor of NAD- glycohydrolase and the microbial toxins.