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）：从预期的蛋白质组学突破来改善人类健康将取决于对蛋白质的展开/折叠和动态的全面了解以及非水溶剂对酶动力学的影响。将从模型辣根过氧化物酶（HRP）的研究中寻求这种理解。将遵循二级和三级结构变化的时间序列，次级和第三级结构的变化以及由非水溶剂诱导的酶活性的变化将相关，并且将研究底物类似物的依基蛋白扩散的动力学。实验方法将包括荧光和磷光，圆形二色性（CD）和紫外线/可见吸光度。最终，将寻求停止流量设备以允许测量更快的分子事件。具体目的是检验五个假设。 1）可以通过监测色氨酸荧光的相对时间顺序，近/紫外CD，远/紫外CD和Soret CD吸光度来确定二级结构和三级结构变化的时间序列。 2）可以测量非水溶液中HRP的构象变化，并与酶活性的显着降低有关。 3）三氟乙醇（TFE）改变了HRP的血红素可及性并改变其酶活性。 4）TFE改变了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通过HRP的底物通道扩散的激活能相关。