Reactivity and Kinetic Properties of TbetaMu Site-Specific Mutants

描述（由申请人提供）：二铜单加氧酶对儿茶酚胺的生物发生至关重要，这种酶调节C-H和氧活化偶联的机制尚不清楚。目前的建议旨在提高对酶如酪胺-单加氧酶（T¿M）激活双氧/C-H键和羟基化底物的机制的理解。这些目标将通过一个可行的重组系统来实现。拥有T¿M重组系统为多巴胺β单加氧酶（D¿M）提供了一个极好的模型，并将提高对昆虫神经传递的理解。此外，T¿M表达系统使产生位点特异性突变更容易，并允许快速和深思熟虑的方法来解决机制问题，因为他们的发展。T¿M可能通过类似于D¿M催化的多巴胺羟基化作用的机制羟基化酪胺。因此，一个三重的研究方法将被应用于检查单个活性位点氨基酸残基的作用。首先，将产生突变体来测试本文所述的Cu位点之间广泛通信的建议。其次，将为每个突变体确定传统的动力学参数（kcat, kcat/Km），并与天然酶进行比较，从而提供关于每个目标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，底物羟基化被认为是通过氢隧穿事件发生的。因此，将使用放射性标记的底物来探索其动力学机制，以确定C-H活化步骤的本征动力学同位素效应（KIE）。突变对氢隧穿的影响将在KIE的温度依赖性中得到体现。多巴胺-单加氧酶（D¿M）控制神经递质多巴胺和去甲肾上腺素的水平。去甲肾上腺素通路异常与高血压、心力衰竭、抑郁症和帕金森病有关。具体来说，D¿M抑制剂已被开发为充血性心力衰竭的潜在治疗方法。帕金森病的主要症状是多巴胺产生和作用不足的结果。使用类似的系统，T¿M，将阐明这一酶家族的催化机制，为我们理解许多神经系统疾病提供分子框架。

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.