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Horseradish Peroxidase: Dynamics and Nonaqueous Activity

辣根过氧化物酶：动力学和非水活性

基本信息

批准号：
6594282
负责人：
MERLYN D. SCHUH
金额：
$ 14.99万
依托单位：
DAVIDSON COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-04-01 至 2006-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6594282
关键词：
atomic absorption spectrometry catalyst circular dichroism conformation enzyme activity enzyme structure enzyme substrate fluorescence high performance liquid chromatography horseradish peroxidase mathematical model phosphorescence protein folding solutions stop flow technique

项目摘要

DESCRIPTION (provided by applicant): Improvements to human health from the anticipated breakthroughs in proteomics will depend on a fuller understanding of unfolding/folding and dynamics of proteins and the effects of nonaqueous solvents on enzyme kinetics. Such understanding will be sought from studies of the model enzyme horseradish peroxidase (HRP). The temporal sequence of changes in secondary and tertiary structure during unfolding will be followed, changes in secondary and tertiary structure and enzyme activity induced by nonaqueous solvents will be correlated, and the dynamics for intraprotein diffusion of substrate analogues will be studied. Experimental methods will include fluorescence and phosphorescence, circular dichroism (CD), and UV/visible absorbance. Eventually, stopped-flow equipment will be sought to permit measurements of faster molecular events. The specific aims are to test five hypotheses. 1) The temporal sequence of changes in secondary structure and tertiary structure during the unfolding of HRP can be determined by monitoring the relative time-course of the tryptophan fluorescence, near/UV CD, far/UV CD, and Soret CD absorbance. 2) Conformational changes in HRP in nonaqueous solutions can be measured and related to the significant reduction of enzyme activity. 3) Trifluoroethanol (TFE) alters the heme accessibility of HRP and alters its enzyme activity. 4) TFE alters the enzyme activity of HRP with other substrates. 5) The structure of substrates and substrate analogues for HRP can be correlated with the activation energy for their diffusion through the substrate channel of HRP.

描述（由申请人提供）：预期的蛋白质组学突破对人类健康的改善将取决于对蛋白质的解折叠/折叠和动力学以及非水溶剂对酶动力学的影响的更全面理解。这种理解将寻求从模型酶辣根过氧化物酶（HRP）的研究。在展开过程中的二级和三级结构的变化的时间顺序将遵循，二级和三级结构的变化和酶活性诱导的非水溶剂将相关，和底物类似物的蛋白质内扩散的动力学将进行研究。实验方法将包括荧光和磷光、圆二色性（CD）和UV/可见光吸光度。最终，将寻求停流设备，以允许更快的分子事件的测量。具体目的是检验五个假设。1)通过监测色氨酸荧光、近/UV CD、远/UV CD和Soret CD吸光度的相对时间过程，可以确定HRP解折叠过程中二级结构和三级结构变化的时间顺序。2)HRP在非水溶液中的构象变化可以被测量并且与酶活性的显著降低相关。3)三氟乙醇（TFE）改变HRP的血红素可及性，并改变其酶活性。4)TFE改变HRP与其他底物的酶活性。5)HRP的底物和底物类似物的结构可以与它们通过HRP的底物通道扩散的活化能相关。