Upgrade of stopped-flow reaction analyzer for enzymatic studies

酶介导大部分基本生命过程。 因此，在分子水平上理解这些过程需要对所涉及的酶进行结构和功能研究。 这种理解也为设计生物技术上有用的催化剂以及新的治疗方法奠定了基础。 了解酶如何工作包括确定它们的机制，即用于将反应物转化为产物的多步反应途径，并阐明其组成氨基酸残基在决定这一功能中的作用。 这两个目标的实现需要适当的动力学分析，涉及瞬态动力学方法。 我们要求对老化的停流（SF）反应分析仪进行必要的升级。 该仪器使得共同申请人能够通过瞬态动力学分析来研究大量酶的机理。 许多这些酶降解污染物，如多氯联苯和N-亚硝基二甲胺。 我们研究的其他酶，如HsaD和Isd血红素摄取系统，对致病细菌至关重要。 我们研究计划的长期目标是了解涉及这些不同蛋白质的生物过程的分子机制。 这些研究还促进了新型生物催化剂和新型抗菌治疗剂的开发。 所要求的软件和硬件包括更换一台过时的计算机和操作系统，这些计算机和操作系统正在失灵，无法升级，同时还包括改进的电子设备、先进的数据分析软件和顺序混合能力。 我们的研究涉及使用互补的方法，包括稳态动力学，诱变和结晶学。 然而，没有瞬态动力学分析，酶的结构-功能研究是不可能的。 SF仪器在UBC是独一无二的，可能在加拿大也是如此，因为它安装在厌氧手套箱中，并且有几个选项来促进我们特定酶的研究。 总的来说，所要求的设备能够进行目前无法进行的实验和分析，并确保SF仪器将在未来几年内使用。

Enzymes mediate the majority of fundamental life processes. An understanding of these processes at the molecular level thus necessitates structural and functional studies of the enzymes involved. Such an understanding also establishes a basis for the design of biotechnologically useful catalysts as well as novel therapeutics. Understanding how enzymes work involves determining their mechanisms, that is, the multiple-step reaction pathway used to transform reactants to products, and elucidating the roles of their constituent amino acid residues in determining this function. The achievement of these two objectives requires proper kinetic analysis involving transient-state kinetic methods. We are requesting an essential upgrade to an aging stopped-flow (SF) reaction analyzer. This instrument enables the co-applicants to study the mechanism of a large number of enzymes by transient-state kinetic analysis. Many of these enzymes degrade pollutants such as PCBs and N-nitrosodimethylamine. Other enzymes we study, such as HsaD and the Isd heme-uptake system, are critical to disease-causing bacteria. The long-term objectives of our research programs are to understand the molecular mechanisms of the biological processes involving these various proteins. These studies also facilitate the development of novel biocatalysts and new antimicrobial therapeutics. The requested software and hardware include replacement of an obsolete computer and operating system that are failing and cannot be upgraded, together with improved electronics, sophisticated data analysis software, and sequential mixing capabilities. Our research involves using complementary approaches including steady-state kinetics, mutagenesis and crystallography. However, proper structure-function studies of enzymes are simply not possible without transient-state kinetic analysis. The SF instrument is unique at UBC, and possibly in Canada, as it is installed in an anaerobic glovebox and has several options to facilitate study of our particular enzymes. Overall, the requested equipment enables experiments and analyses not currently possible and ensures that the SF instrument will be used for years to come.