Reactivity and Kinetic Properties of TbetaMu Site-Specific Mutants

描述(由申请人提供)：双加氧酶对儿茶酚胺的生物发生至关重要，这种酶调节C-H偶联和氧激活的机制尚不清楚。目前的提议旨在提高对酪胺β-单加氧酶(T？M)等酶激活氧/C-H键和羟化底物这一机制的理解。这些目标将使用可用于T？M的可行的重组系统来实现。拥有T？M重组系统将为多巴胺β-单加氧酶(D？M)提供一个极好的模型，并将提高对昆虫神经传递的理解。此外，T？M表达系统使产生特定位点的突变体变得更容易，并允许快速和深思熟虑地解决发展中的机械性问题。T？M可能以类似于D？M催化的多巴胺羟基化的机制羟化酪胺。因此，将采用三重研究方法来研究单个活性部位氨基酸残基的作用。首先，将产生突变体来测试在此描述的铜位置之间的通信广泛的提议。其次，将为每个突变体确定传统的动力学参数(kcat，kcat/Km)，并将其与天然酶进行比较，从而提供关于每个靶向残基如何参与电子转移(在kcat中检测到)或底物激活(在kcat/Km中检测到)的重要信息。第三，底物羟化被认为是通过氢隧道事件进行的。因此，将利用放射性标记底物来确定C-H活化步骤的本征动力学同位素效应(Kie)，以探讨其动力学机制。我们将研究突变对氢隧穿的影响，这将在KIE的温度依赖关系中表现出来。与公众健康相关这种酶，即多巴胺β-单加氧酶(D？M)，控制着神经递质、多巴胺和去甲肾上腺素的水平。去甲肾上腺素途径的异常与高血压、心力衰竭、抑郁症和帕金森氏症有关。具体地说，D？M抑制剂已被开发为治疗充血性心力衰竭的潜在药物。帕金森氏症的主要症状是多巴胺的生成和作用不足。使用一个类似的系统，T？M，将阐明这一酶家族的催化机制，为我们理解许多神经疾病提供分子框架。

DESCRIPTION (provided by applicant): Dicopper monooxygenases are critical for the biogenesis of catecholamines, and the mechanism by which such enzymes regulate the coupling of C-H and oxygen activation remains unclear. The current proposal aims to improve the understanding of this mechanism by which enzymes like tyramine beta monooxygenase (T¿M) activate dioxygen/C-H bonds and hydroxylate substrates. The objectives will be accomplished using a workable recombinant system available for T¿M. Having a T¿M recombinant system provides an excellent model for dopamine beta monooxygenase (D¿M) and will improve the understanding of neurotransmission in insects. In addition, the T¿M expression system makes generating site-specific mutants easier and allows a quick and deliberate approach to address mechanistic questions as they develop. T¿M likely hydroxylates tyramine by a mechanism similar to dopamine hydroxylation catalyzed by D¿M. Consequently, a three-fold research approach will be applied to examine the role of individual active site amino acid residues. First, mutants will be generated to test the proposal described herein that communication between the Cu sites is extensive. Second, traditional kinetic parameters (kcat, kcat/Km) will be determined for each mutant and compared to the native enzyme, thus providing important information on how each targeted residue is involved with either electron transfer (detected in kcat) or substrate activation (detected in kcat/Km). Third, substrate hydroxylation is proposed to take place by a hydrogen tunneling event. Thus, the kinetic mechanism will be probed using radioactive labeled substrates to determine the intrinsic kinetic isotope effect (KIE) on the C-H activation step. The effect of mutation on hydrogen tunneling will be examined and this will be manifest in the temperature dependence of the KIE. PUBLIC HEALTH RELEVANCE The enzyme, dopamine beta-monooxygenase (D¿M), controls the levels of the neurotransmitters, dopamine and norepinephrine. Irregularities in the norepinephrine pathway are linked to hypertension, heart failure, depression, and Parkinson's disease. Specifically, D¿M inhibitors have been developed as a potential treatment of congestive heart failure. The primary symptoms of Parkinson's disease are the result of insufficient generation and action of dopamine. Using an analogous system, T¿M, will clarify the mechanism of catalysis for this family of enzymes providing the molecular framework for our understanding of a number of neurological diseases.