Chemical Mechanisms of Biosynthesis

生物合成的化学机制

基本信息

批准号：
7636843
负责人：
RICHARD VANCE WOLFENDEN
金额：
$ 39.19万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1980
资助国家：
美国
起止时间：
1980-07-01 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7636843
关键词：
Acceleration Acetates Active Sites Affinity Alanine Amines Anabolism Anti-Bacterial Agents Antimalarials Arginine Aspartate Autoimmune Diseases Binding Biological Models Carbamoyl Transferases Carbon Dioxide Carboxy-Lyases Catalysis Cell division Chemicals Chlorophyll Commit Complex Cytochromes Decarboxylation Disease Electrostatics Entropy Environment Enzymes Half-Life Heating Homoserine Human Kinetics Laboratories Learning Lysine Malignant Neoplasms Measurement Metals Methionine Methods Minor Modeling N-Acetylglucosamine kinase Normal Cell Nucleotide Biosynthesis Organism Orotidine-5&apos -Phosphate Decarboxylase Pathway interactions Patients Peptidyltransferase Play Porphyrias Predisposition Property Protons Pyrimidine Nucleotides Pyrroles Reaction Reporting Ribosomes Role Sequence Homology Side Stretching Temperature Testing Therapeutic Thermodynamics Uroporphyrinogen Decarboxylase Uroporphyrinogens Ursidae Family Water Work analog base carbanion catalyst cofactor design enzyme substrate complex functional group heme biosynthesis hexokinase improved inhibitor/antagonist mutant public health relevance reaction rate research study uroporphyrin III

项目摘要

DESCRIPTION (provided by applicant): In cancer and autoimmune diseases, rapid cell division depends on nucleotide biosynthesis rather than the salvage pathways that predominate in normal cells. Thus, mechanism-based inhibitors of orotidine 5-phosphate decarboxylase (ODCase), the last step in the formation of pyrimidine nucleotides, may have therapeutic value in the treatment of these diseases, and as antibacterial and antimalarial agents. We will use kinetic and mass spectrometric measurements to test the possibility that the imperfect binding of the transition state analogue 6-hydroxy-UMP arises from the trapping of a water molecule in its inhibitory complex with ODCase, in a gap deep in the active site that is believed to be occupied by CO2 released during decarboxylation. Based on recent observations of the binding affinities of 6-C(=S)NH2 and 6-C(=Se)NH2 derivatives of UMP, we will also prepare new 6-substituted inhibitors that are expected to fill that gap and bear a closer resemblance to the altered substrate in the transition state, in which the C-C bond is stretched but not broken. Preliminary experiments in this laboratory indicate that uroporphyrin III decarboxylase (UroD), which is missing in most patients with porphyria of the most common type, generates one of the largest rate enhancement that has been observed for any enzyme. Unlike ODCase, UroD is almost devoid of active site residues that might be involved in catalysis. We propose to test the possibility that 2 essential arginine residues play a direct role in catalysis, by stabilizing the carbanion generated by the departure of CO2, then furnishing the proton that takes its place; and that this reaction represents an extreme example of catalysis by desolvation. These analogues are expected to be useful, in conjunction with exact structural methods, in working out the detailed mechanism of catalysis by this enzyme. Model experiments will also be conducted to determine the susceptibility of decarboxylation to catalysis by desolvation, and to catalysis by molecules representing groups that are present at the human enzyme's active site. PUBLIC HEALTH RELEVANCE: In cancer and autoimmune diseases, rapid cell division depends on nucleotide biosynthesis rather than the salvage pathways that predominate in normal cells. This project involves the design of mechanism-based inhibitors of orotidine 5- phosphate decarboxylase (ODCase), the last step in the formation of pyrimidine nucleotides, may have therapeutic value in the treatment of these diseases, and as antibacterial and antimalarial agents. This project also aims to establish the mechanism of action of uroporphyrin III decarboxylase, whose deficiency is responsible for the most common form of porphyria. There is reason to believe that this reaction, which occurs extremely slowly in the absence of enzyme (half- life 25,000 years) may represent an extreme example of catalysis by desolvation; if that is found to be the case, then there are reasonable prospects of developing artificial catalysts to alleviate the condition.

描述（由申请人提供）：在癌症和自身免疫性疾病中，快速细胞分裂取决于核苷酸生物合成，而不是在正常细胞中占主导地位的挽救途径。因此，基于机理的5-磷酸脱羧酶（ODCase）的基于机理的抑制剂（ODCase）是嘧啶核苷酸形成的最后一步，在治疗这些疾病的治疗中可能具有治疗价值，作为抗菌和抗菌药物。我们将使用动力学和质谱测量测量来测试过渡状态模拟6-羟基的不完善结合是由于水分子在其抑制性复合物中与ODCase的捕获在其抑制性络合物中的捕获，而在脱键式羧基化过程中释放的CO2占据的活性位点的间隙深处。基于对UMP的6-C（= S）NH2和6-C（= SE）NH2衍生物的结合亲和力的最新观察，我们还将准备新的6个6个取代的抑制剂，这些抑制剂有望填补该空白并具有与C-C键的变化底物的相似之处，而C-C键则在C-C键中均未伸展。该实验室中的初步实验表明，在大多数最常见类型的斑岩患者中缺少尿磷脂III脱羧酶（UROD），这是任何酶观察到的最大率提高之一。与ODCASE不同，UROD几乎没有可能参与催化的活性位点残基。我们建议通过稳定二氧化碳出发产生的碳纤维，然后提供取代其代替的质子来测试2种基本精氨酸残基在催化中发挥直接作用的可能性；并且这种反应代表了通过脱溶作催化的极端例子。预计这些类似物将与精确的结构方法结合使用，以确定该酶催化的详细机制。还将进行模型实验，以确定脱羧基对脱溶作催化的敏感性，以及对人类活性位点中存在的组的分子进行催化。公共卫生相关性：在癌症和自身免疫性疾病中，快速细胞分裂取决于核苷酸生物合成，而不是占主导地位的挽救途径。该项目涉及设计基于机制的5-磷酸脱羧酶（ODCASE）的基于机制的抑制剂，这是嘧啶核苷酸形成的最后一步，可能在治疗这些疾病的治疗中具有治疗价值，并作为抗体和抗体和抗微药物具有治疗价值。该项目还旨在建立泌尿核酸III脱羧酶的作用机理，其缺乏症是最常见的卟啉症形式。有理由相信，这种反应在没有酶的情况下非常缓慢地发生（半人生25,000年）可能代表了脱溶作催化的极端例子。如果发现这种情况，那么有合理的前景是开发人工催化剂以减轻这种情况。