Upgrade of stopped-flow reaction analyzer for enzymatic studies

点击翻译按钮获取中文摘要

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.