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NEW CHEMICAL APPROACHES TO ADP-RIBOSYL TRANSFER

ADP-核糖基转移的新化学方法

基本信息

批准号：
3281966
负责人：
JAMES T SLAMA
金额：
$ 8.11万
依托单位：
UNIVERSITY OF TOLEDO
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-12-01 至 1994-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3281966
关键词：
ADP ribosylation NAD nucleosidase NAD(H) analog chemical binding cholera toxin diphtheria toxin enzyme inhibitors enzyme mechanism enzyme structure enzyme substrate analog nucleic acid chemical synthesis nucleotide analog pertussis toxin posttranslational modifications protein purification

项目摘要

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.

ADP-核糖基转移酶是一类重要的调节酶。该项目提出了NAD特异性的设计和亲和标记 ADP-核糖基转移酶的底物结合位点。的信息所寻求的是鉴定催化必需氨基酸残基和活性位点结构的阐明。鉴定必需氨基酸残基和活性位点结构的阐明。必需氨基酸残基的鉴定将促进了解这些独特催化剂的机制。这信息将支持基因工程的发展针对百日咳和相关疾病的疫苗，其中微生物ADP- 核糖基转移酶作为外毒素组分起作用。亲和性和光亲和性标记将被应用于本发明的研究。哺乳动物NAD糖水解酶作为水解转移酶的实例和催化活性。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，作为单（ADP-核糖基）类的代表是重要的转移酶已被牵连作为一个新的细胞调节机制亲和标记和光亲和标记均不特异于 NAD底物结合位点，并且通常适用于类目前可用。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切割的 NAD类似物。第一种生产亲和标记的方法和光亲和标记因此成为可能，我们的发展，不可切割的类似物。第一种生产方法亲和标记和光亲和标记因此通过以下步骤成为可能：我们开发的不可裂解的类似物卡巴-NAD。这里环戊烷环取代烟酰胺的β-D-核苷酸 NAD的核苷部分。已证明Carba-NAD对ADP-核糖基转移具有抗性并且是NAD糖水解酶的有效竞争性抑制剂，几种单（ADP-核糖基）转移酶。有亲缘关系的家庭标记和光亲和标记将通过修饰 carba-NAD，并应用于上述酶的研究。一抑制剂设计的第二种策略将利用氨基糖作为 "过渡态类似物"来模拟氧碳正离子中间体提出了对Nacaliam NAD糖水解酶。1，4-二甲氧基苯甲酸的合成双脱氧-4-氨基-D-呋喃核糖是可获得的，将氨基糖转化为ADP-核糖类似物将容易实现。的 "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。