Chemical Mechanisms of Biosynthesis

生物合成的化学机制

基本信息

批准号：
7527666
负责人：
RICHARD VANCE WOLFENDEN
金额：
$ 37.93万
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/7527666
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 Condition Cytochromes Decarboxylation Depth 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 Object Attachment Organism Orotidine-5&apos -Phosphate Decarboxylase Pathway interactions Patients Peptidyltransferase Play Porphyrias Predisposition Property Protons Public Health Pyrimidine Nucleotides Pyrroles Rate Reaction Reporting Ribosomes Role Sequence Homology Side Stretching Temperature Testing Therapeutic Thermodynamics Uridine Monophosphate 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 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，而不是正常细胞中占主导地位的挽救途径。因此，基于机制的乳清苷5-磷酸脱羧酶（ODCase）抑制剂（嘧啶核苷酸形成的最后一步）可能在这些疾病的治疗中具有治疗价值，并可作为抗菌剂和抗疟剂。我们将使用动力学和质谱测量来测试过渡态类似物 6-羟基-UMP 的不完美结合是否是由于水分子被捕获在其与 ODCase 的抑制复合物中而产生的可能性，该复合物位于活性位点深处的间隙中，据信被脱羧过程中释放的CO2占据。基于最近对 UMP 的 6-C(=S)NH2 和 6-C(=Se)NH2 衍生物的结合亲和力的观察，我们还将制备新的 6-取代抑制剂，有望填补这一空白并具有更紧密的联系。类似于过渡态下改变的基底，其中 C-C 键被拉伸但没有断裂。该实验室的初步实验表明，尿卟啉 III 脱羧酶 (UroD) 在大多数最常见类型的卟啉症患者中缺失，但它产生的速率增强是已观察到的任何酶中最大的速率增强之一。与 ODCase 不同，UroD 几乎不含可能参与催化的活性位点残基。我们建议测试 2 个必需的精氨酸残基在催化中发挥直接作用的可能性，通过稳定 CO2 离开时产生的碳负离子，然后提供取代其位置的质子；该反应代表了去溶剂化催化的一个极端例子。这些类似物与精确的结构方法相结合，预计可用于研究该酶催化的详细机制。还将进行模型实验以确定脱羧对去溶剂化催化的敏感性，以及对代表存在于人类酶活性位点的基团的分子催化的敏感性。公共健康相关性：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，而不是正常细胞中占主导地位的挽救途径。该项目涉及基于机制的乳清苷5-磷酸脱羧酶（ODCase）抑制剂的设计，ODCase是嘧啶核苷酸形成的最后一步，可能在这些疾病的治疗中具有治疗价值，并可作为抗菌和抗疟药。该项目还旨在建立尿卟啉 III 脱羧酶的作用机制，该酶的缺乏是导致最常见形式的卟啉症的原因。有理由相信，这种在没有酶存在的情况下发生得极其缓慢的反应（半衰期为 25,000 年）可能代表了去溶剂化催化的一个极端例子。如果情况确实如此，那么开发人工催化剂来缓解这种情况就有合理的前景。