STRUCTURAL MODELS FOR MOLYBDOENZYMES

钼酶的结构模型

• 批准号: 6726569
• 负责人: JOHN H ENEMARK
• 金额: $ 30.48万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-06-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6726569
• 关键词: active sites; biophysics; catalyst; circular magnetic dichroism; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; electron transport; flash photolysis; metalloenzyme; model design /development; molybdenum; mutant; oxidation reduction reaction; oxygen; oxygen transport; physical model; sulfites; technology /technique development

Molybdenum is an essential trace element for all forms of life, and over 50 molybdoenzymes are known that catalyze oxidation-reduction reactions that are essential in the metabolism of carbon, nitrogen and sulfur. In humans molybdoenzymes play physiologically vital roles in the oxidation of sulfite to sulfate (sulfite oxidase) and in certain aspects of purine metabolism (xanthine oxidase, xanthine dehydrogenase). Fatal simultaneous deficiencies in the activities of these enzymes due to an inborn deficiency of a "molybdenum cofactor" have been well documented in children. Point defects in the sulfite oxidase protein itself can also produce the severe neurological symptoms of sulfite oxidase deficiency. These symptoms include dislocated ocular lenses, mental retardation, and, in severe cases, early death. Development of methods to clone and express human sulfite oxidase has revealed several different clinical point mutations that result in isolated sulfite oxidase deficiency. The X-ray structure of the highly homologous chicken liver sulfite oxidase provides a molecular basis for interpreting the fatal point mutations of the human enzyme. The research proposed here addresses the fundamental properties of sulfite oxidase and other molybdoenzymes by an integrated program of biophysical, biochemical and model compound studies. Emphasis will be given to the use of variable frequency pulsed electron paramagnetic resonance (EPR) techniques, especially electron spin echo envelope modulation (ESEEM) and pulsed electron-nuclear double resonance (ENDOR) spectroscopies, to probe in detail the surroundings of the molybdenum active site in wild-type and mutant enzymes. The trafficking of oxygen atoms at the molybdenum center during catalysis will be followed by pulsed EPR studies in water enriched in 17-O. The electron transfer reactions at the molybdenum center will be investigated by protein film voltammetry, and the factors that control the rates of intramolecular electron transfer between the molybdenum and iron center in native and mutant sulfite oxidase will be investigated by flash photolysis and theoretical modeling.

钼是所有生命形式必需的微量元素，已知有超过 50 种钼酶可催化氧化还原反应，这些反应对于碳、氮和硫的代谢至关重要。在人体中，钼酶在亚硫酸盐氧化为硫酸盐（亚硫酸盐氧化酶）以及嘌呤代谢的某些方面（黄嘌呤氧化酶、黄嘌呤脱​​氢酶）中发挥着重要的生理作用。在儿童中，由于先天性缺乏“钼辅因子”而导致这些酶的活性同时发生致命性缺陷，这一情况已有充分记录。亚硫酸氧化酶蛋白本身的点缺陷也会产生亚硫酸氧化酶缺乏的严重神经症状。这些症状包括晶状体脱位、智力低下，严重时甚至会过早死亡。克隆和表达人亚硫酸盐氧化酶方法的开发揭示了几种不同的临床点突变，这些突变导致孤立的亚硫酸盐氧化酶缺陷。高度同源的鸡肝亚硫酸氧化酶的X射线结构为解释人类酶的致命点突变提供了分子基础。这里提出的研究通过生物物理、生物化学和模型化合物研究的综合计划解决了亚硫酸氧化酶和其他钼酶的基本特性。重点将强调使用变频脉冲电子顺磁共振（EPR）技术，特别是电子自旋回波包络调制（ESEEM）和脉冲电子核双共振（ENDOR）光谱，以详细探测钼活性物质的周围环境野生型和突变型酶中的位点。催化过程中氧原子在钼中心的迁移随后将在富含 17-O 的水中进行脉冲 EPR 研究。将通过蛋白质膜伏安法研究钼中心的电子转移反应，并通过闪光光解和理论模型研究控制天然和突变亚硫酸盐氧化酶中钼和铁中心之间分子内电子转移速率的因素。