DESIGN OF MECHANISM BASED INHIBITORS OF DNA SYNTHESIS

基于机制的 DNA 合成抑制剂的设计

• 批准号: 3072325
• 负责人: JOANNE STUBBE
• 金额: $ 6.01万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-10-01 至 1988-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3072325
• 关键词: Escherichia coli; affinity chromatography; allosteric site; amides; aminoacyltransferase; antibody; chemical synthesis; chickens; electron spin resonance spectroscopy; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; glutamine; glycine; iron; ligase; liver; oxidoreductase; purines; ribonucleotides; stop flow technique

This proposal is concerned with a study of the mechanism of two enzymes involved in DNA synthesis and use of this mechanistic information to design specific inactivators of these enzymes. The enzymes are ribonucleotide reductase (RDPR) and formylglycinamidine ribonucleotide (FGAM) synthetase. 1. Ribonucleotide reductases are uniquely responsible for the reduction of nucleotides to deoxynucleotides and have been divided into classes defined by the nature of their cofactor. This proposal is concerned with the class of reductases represented by the E. coli enzyme which requires 2 Fe+3 and an organic tyrosyl radical for activity. We have proposed a new mechanism for this reduction involving a radical cation intermediate. The basic objectives are: (1) to obtain evidence for, or against, a homolytic mechanism using rapid quench EPR spectroscopy and rapid scan spectroscopy; (2) to study the nature of the active site using the unique classes of suicide inhibitors; (3)to design several new classes of RDPR suicide inhibitors based on mechanistic information. 2. FGAM Synthetase is an enzyme involved in the early stages of purine biosynthesis. The basic objectives are (1) to examine in detail the mechanism of conversion of formyglycineamide ribonucleotide (FGAR) to FGAM in order to obtain evidence for, or against, chemically and kinetically competent phospho-E or phospho-FGAR intermediates; (2) To examine the substrate specificity of FGAM synthetase and to use this information in conjunction with the mechanistic information to design potent specific inhibitors; (3) to prepare antibodies to native enzyme for use in determination of this enzyme's concentration in crude cell extracts and to determine if this antibody is cross-reactive with a larger molecular weight peptide in crude cells which may possess enzymatic activity of other early enzymes in the purine biosynthetic pathway; (4) to utilize FGAR amidotransferase and the antibody prepared to it as potential affinity columns in the isolation of phosphoribosylamine (PRA) amidotransferase. Our long range goals involve isolation of the first five enzymes in the purine biosynthetic pathway and eventual reconstitution of these enzymes in an attempt to find evidence, kinetic and biophysical for a functional association.

这一建议涉及两种酶的作用机制的研究 参与DNA合成，并利用这些机械信息来设计 这些酶的特异性灭活剂。 这些酶是核糖核苷酸 还原酶（RDPR）和甲酰甘氨脒核糖核苷酸（FGAM）合成酶。 1.核糖核苷酸还原酶是唯一负责还原 核苷酸到脱氧核苷酸，并已被分为定义的类别 由其辅因子的性质决定。 这个建议与班级有关 以E.大肠杆菌酶，需要2 Fe+3， 活性的有机酪氨酰基自由基。 我们提出了一个新的机制 因为这种还原涉及自由基阳离子中间体。 基本目标是：（1）获得支持或反对某项指控的证据。 用快速淬灭EPR谱和快速扫描研究均裂机理 光谱;（2）研究活性位点的性质，使用独特的 （3）设计几类新的RDPR 基于机械信息的自杀抑制剂。 2. FGAM合成酶是 一种参与嘌呤生物合成早期阶段的酶。 基本 目标是（1）详细研究 甲酰甘氨酸酰胺核苷酸（FGAR）到FGAM，以获得证据 对于，或针对，化学和动力学上有能力的磷酸-E或 磷酸-FGAR中间体;（2）检查底物特异性 FGAM合成酶，并将此信息与 设计有效的特异性抑制剂的机制信息;（3） 制备天然酶的抗体，用于测定 酶的浓度在粗细胞提取物，并确定这是否 抗体与粗品中的较大分子量肽交叉反应 细胞中可能具有其他早期酶的酶活性， 嘌呤生物合成途径;（4）利用FGAR酰胺转移酶和 制备的抗体作为潜在的亲和柱， 磷酸核糖胺（PRA）酰胺转移酶。 我们的长期目标包括 分离嘌呤生物合成途径中的前五种酶， 最终重组这些酶，试图找到证据， 动力学和生物物理学的功能关联。