Structure and Enzyme Function in Glyoxylate Metabolism and Hyperoxaluria

乙醛酸代谢和高草酸尿中的结构和酶功能

• 批准号: 7079651
• 负责人: W TODD LOWTHER
• 金额: $ 20.44万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7079651
• 关键词: NAD(H) phosphate; X ray crystallography; active sites; alcohol oxidoreductases; chemical kinetics; cofactor; enzyme activity; enzyme complex; enzyme structure; enzyme substrate; fatty acid metabolism; gene mutation; human genetic material tag; molecular pathology; nephrocalcinosis; oxalates; primary hyperoxalurias; protein binding; protein structure function; pyruvates; short chain fatty acid; structural biology

DESCRIPTION (provided by applicant): This R21 proposal will investigate the structure-function relationships of human glyoxylate/hydroxypyruvate reductase (GRHPR), a key enzyme in glyoxylate and hydroxypyruvate metabolism. Defects in human GRHPR are present in the rare genetic disease primary hyperoxaluria type 2 (PH2). These mutations ultimately result in the buildup of oxalate and the formation and deposition of urinary tract calcium oxalate kidney stones. An altered GRHPR activity could also contribute to idiopathic stone disease, a common debilitating health problem that impacts daily life and incurs significant health care costs. The historical analysis of GRHPR from other organisms has yielded contradictory evidence for the preference of cofactor, the salt dependence of the reaction, and substrate inhibition. In addition, no structural or biochemical experiments have been reported for human GRHPR. The long-term goals of this research are to characterize the kinetic properties of human GRHPR, to identify the structural features that determine its activity, and to understand how these properties are altered in mutant enzymes causing PH2. The proposed study will (Aim 1) determine the crystal structures of human GRHPR alone and in complex with NADPH and (Aim 2) determine the cofactor and substrate specificity of human GRHPR through the biochemical analysis of wild-type and PH2 mutant enzymes. The structures of human GRHPR will enable the mapping of current and future PH2 variants onto the structure and the prediction of the physiological consequences. The biochemical data will reveal the kinetic parameters associated with the interaction of the enzyme with substrates, cofactors and modulating anions. The cha racterization of human GRHPR will help establish a more precise physiological role for the enzyme and help explain how mutations cause disease. Such studies may ultimately lead to improved treatment strategies for individulas with PH2 and possibly those with idiopathic stone disease.

描述（由申请人提供）：该R21提案将研究人甘酰胺/羟基丙酮酸还原酶（GRHPR）的结构 - 功能关系，这是一种甘酰基和羟基型型型型型型型代谢中的关键酶。人类GRHPR缺陷存在于罕见的遗传疾病原发性高黄油2型（PH2）中。这些突变最终导致草酸盐的积聚以及草酸钙钙肾结石的形成和沉积。 GRHPR活动的改变也可能导致特发性石材疾病，这是一个常见的使人衰弱的健康问题，会影响日常生活并造成巨大的医疗保健成本。其他生物体对GRHPR的历史分析为辅助因子的偏好，反应的盐依赖性和底物抑制提供了矛盾的证据。另外，尚无人Grhpr的结构或生化实验。这项研究的长期目标是表征人类GRHPR的动力学特性，以确定确定其活性的结构特征，并了解这些特性如何改变引起PH2的突变酶中。拟议的研究将（AIM 1）通过野生型和pH2突变酶的生化分析来确定人类GRHPR的晶体结构，并与NADPH和（AIM 2）确定人GRHPR的辅因子和底物特异性。人类GRHPR的结构将使当前和未来的PH2变体能够映射到结构和生理后果的预测。生化数据将揭示与酶与底物，辅因子和调节阴离子相互作用相关的动力学参数。人类GRHPR的CHA竞争将有助于建立更精确的生理作用，并有助于解释突变如何引起疾病。此类研究最终可能会改善使用PH2以及可能患有特发性石材疾病的个体的治疗策略。