PROPERTIES OF AORTIC LYSYL OXIDASE

弹性蛋白和胶原蛋白的生物合成涉及翻译后， 每种新合成的蛋白质的酶催化加工。 在这些基本过程中，肽基赖氨酸的氧化， 由赖氨酰氧化酶催化，启动形成内， 分子间赖氨酸衍生交联，其稳定和 不溶解这些结缔组织蛋白的细胞外纤维。 由于这似乎是纤维合成的最后一步， 这可能是生物防治的关键点， 结缔组织 此外，赖氨酰氧化酶是一种合理的 抗纤维化剂的化疗靶点， 限制肺纤维化中纤维化后遗症的发展， 动脉粥样硬化和限制疤痕形成。 现时的建议 旨在定义作用机制和底物的关键方面， 赖氨酰氧化酶的特异性，并基于这些信息，设计， 在试管和组织水平上合成和测试新的、机制- 基于肽基和非肽基的这种交联酶的抑制剂。 这些努力将涉及分析吡咯并喹啉的作用， 醌（PQQ）和铜离子辅因子，采用电子 顺磁共振、吸收和荧光分光光度法， 还原和氧化半的停流动力学分析 由这种酶催化的反应。 酶的特异性将是 通过固体组装的寡肽底物的动力学分析来探测 相肽合成。 活性位点的肽环境将 通过氨基酸序列分析和质谱法评估 从酶中分离的肽用共价、放射性标记， 活性位点的探针。 抑制的化学和动力学机制 通过针对辅因子或活性位点亲核试剂的新抑制剂 将被分析。 研究了这种新型抑制剂对交联反应的影响， 将在器官培养中评估鸡胚盲肠中弹性蛋白的含量， 其作为体内抗纤维化剂潜力的指数。

The biosynthesis of elastin and collagen involves post-translational, enzyme-catalyzed processing of each of the newly synthesized proteins. Among these essential processes, the oxidation of peptidyl lysine, catalyzed by lysyl oxidase, initiates the formation of intra- and intermolecular lysine-derived crosslinkages which stabilize and insolubilize extracellular fibers of these connective tissue proteins. Since this appears to be the last step in fiber synthesis, it is potentially a key point of biological control in the development of connective tissues. Moreover, lysyl oxidase is a rational chemotherapeutic target of anti-fibrotic agents designed to prevent of limit the development of fibrotic sequellae as in lung fibrosis and atherosclerosis and to limit scar formation. The present proposal intends to define key aspects of the mechanism of action and substrate specificity of lysyl oxidase and, based on such information, to design, synthesize and test at both test-tube and tissue levels new, mechanism- based, peptidyl and non-peptidyl inhibitors of this crosslinking enzyme. These efforts will involve analyses of the roles of the pyrroloquinoline quinone (PQQ) and copper ion cofactors, employing techniques as electron paramagnetic resonance, absorption and fluorescence spectrophotometry, and stopped flow kinetic analyses of the reductive and oxidative half reactions catalyzed by this enzyme. Specificity of the enzyme will be probed by kinetic analyses of oligopeptide substrates assembled by solid phase peptide synthesis. The peptide environment of the active site will be assessed by amino acid sequence analysis and mass spectroscopy of peptides isolated from the enzyme labelled with covalent, radioactive probes of the active site. Chemical and kinetic mechanisms of inhibition by new inhibitors directed at the cofactor or active site nucleophiles will be analyzed. The effect of such new inhibitors on the crosslinking of elastin in chick embryo aortae will be assessed in organ culture as an index of their potential as anti-fibrotic agents in vivo.