STRUCTURE-ACTIVITY RELATIONSHIP STUDIES OF DOPAMINE BETA-MONOOXYGENASE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 铜酶，多巴胺β-单加氧酶（DbM; E.C. 1.14.17.1）在儿茶酚胺神经递质生物合成中起中心作用。因此，在过去的五十年中，DbM的化学和动力学机制得到了广泛的研究。然而，由于缺乏酶活性位点的结构和分子细节，实验结果与D-β-M结构参数的相关性滞后。拟定研究的总体目标是在适用于定点诱变研究的系统中表达人D-β-M，并使用纯化的重组野生型和突变蛋白进行系统的生物化学、生物物理学、结构和机制研究。 长期以来，由于D-β-M在神经递质NE的生物合成中的中心作用，D-β-M一直被认为是用于治疗目的的交感神经系统活性调节的重要靶标。例如，交感神经系统及其神经递质NE在高血压和充血性心力衰竭的发展和维持中的重要作用已被广泛研究。最近的研究还表明，DbM活性在体内的调节可能对可卡因成瘾的治疗具有有益的作用。因此，在分子水平上更好地理解DbM的结构-活性关系对于确定这些疾病的病因和最终开发有效的治疗方法将是重要的。此外，DbM是一个相对更具体的非血红素单加氧酶的一大群的原型，其催化机制的细节可以扩展到其他类似酶的分子机制的理解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The copper enzyme, dopamine beta-monooxygenase (DbM; E.C. 1.14.17.1) plays a central role in catecholamine neurotransmitter biosynthesis. Consequently, the chemical and the kinetic mechanisms of DbM have been extensively studied during the last five decades. However, the correlation of the experimental findings with the structural parameters of D-beta-M is lagging behind due to the lack of structural and molecular details of the active site of the enzyme. The overall objective of the proposed studies is to express human D-beta-M in a system suitable for site-directed mutagenesis studies and to carry out systematic biochemical, biophysical, and structural and mechanistic studies using purified recombinant wild type and mutant proteins. D-beta-M has long been recognized as an important target for the modulation of sympathetic nervous system activity for therapeutic purposes due to its central role in the biosynthesis of the neurotransmitter, NE. For example, the vital role of the sympathetic nervous system and its neurotransmitter NE in the development and maintenance of hypertension and congestive heart failure have been extensively studied. Recent studies have also shown that the regulation of DbM activity in vivo may have beneficial effects on the treatment of cocaine addiction. Therefore, better understanding of the structure-activity relationship of DbM at the molecular level will be important in determining the etiology of these diseases and eventual development of effective therapeutics. In addition, DbM is prototypical of a large group of relatively more specific non-heme monooxygenases and the details of its catalytic mechanism could be extended toward the understanding of the molecular mechanisms of other similar enzymes.