Browsing by Subject "Chemical mechanism"
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Item Mechanistic studies of two enzymes that employ common coenzymes in uncommon ways(2010-08) Thibodeaux, Christopher James; Liu, Hung-wen, 1952-Enzymes are biological catalysts which greatly accelerate the rates of chemical reactions, oftentimes by many orders of magnitude over the uncatalyzed reaction. The remarkable catalytic rate enhancement afforded by enzymes derives ultimately from the structure and chemical properties of the enzyme active sites, which allow enzymes to selectively bind to their substrates and to stabilize high energy chemical species and unstable intermediates along the reaction coordinate. To enhance their catalytic ability, many enzymes have also evolved to require coenzymes for optimal activity. These coenzymes often provide chemical functionality and reactivity that are not accessible by the twenty canonical amino acids and, hence, coenzymes serve to greatly enhance the diversity of chemical reactions that can be mediated by enzymes. The work described in this dissertation focuses on mechanistic studies of two enzymes that use common coenzymes in unusual ways. In the first section of this work, studies will focus on the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2), an essential enzyme in isoprenoid biosynthesis that employs a flavin mononucleotide (FMN) coenzyme for catalysis. In most biological systems, flavin coenzymes mediate electron transfer reactions. However, the IDI-2 catalyzed reaction involves no net redox change, raising questions as to the role of the flavin in the chemical mechanism. The chemical mechanism of IDI-2 will be interrogated with a combination of spectroscopic studies and biochemical techniques. Our studies suggest that the flavin coenzyme of IDI-2 may employ a novel mode of flavin-dependent catalysis involving acid/base chemistry. In the second section of this dissertation, attention will be focused on elucidating the chemical mechanism of 1-aminocyclopropane-1-carboxylate deaminase (ACCD), an enzyme that plays a role in regulating the production of the potent plant hormone, ethylene. ACCD is a pyridoxal-5ʹ-phosphate (PLP)-dependent enzyme that catalyzes a C-C bond cleavage event that is unique among the catalytic cycles of PLP-dependent enzymes. Altogether, our mechanistic studies of IDI-2 and ACCD help to illustrate the catalytic diversity of common coenzymes, and demonstrate that some enzymes have evolved to exploit readily available coenzymes for atypical reactions.Item Synthetic approaches to investigate the chemical mechanism in the biosynthesis of natural products(2012-08) Choi, Sei Hyun; Liu, Hung-wen, 1952-The study of the biosynthetic logic of natural products has established itself to be one of the more exciting areas of research and have become an important part of modern drug discovery and development efforts. Therefore, understanding the pathway and the chemical mechanism of the biosynthesis of natural products is important in that knowledge on these processes can be applied for combinatorial biosynthesis to generate new natural product derivatives with enhanced biological activities. In addition to the practical value, a lot of unprecedented chemical mechanisms can be found in the enzymes involved therein, which will significantly advance our understanding of enzyme catalysis. The works described in this dissertation focus on elucidating the chemical mechanism of a number of enzymes involved in natural product biosynthesis by utilizing the versatility of synthetic chemistry to prepare enzyme substrates and mechanistic probes. First, SpnF and SpnL responsible for constructing the tetracyclic architecture of spinosyn A have been investigated. In vitro assay revealed the importance of the highly conjugated system for the [4+2]cycloaddition catalyzed by SpnF. Biochemical studies strongly suggest that SpnL employs the Rauhut-Currier mechanism for the second cyclization step in the biosynthesis of spinosyn A. It was also demonstrated that SpnL requires SAM for its activity. Second, a radical SAM enzyme DesII involved in the desosamine pathway has been investigated. It has been demonstrated that DesII can catalyze the dehydrogenation of TDP-D-quinovose as well as the deamination of the natural substrate, which makes DesII unique among radical SAM enzymes. In vitro assays revealed that DesII requires stoichiometric amount of SAM, which. EPR study firmly established the intermediacy of a C-3 radical in the DesII-catalyzed dehydrogenation of TDP-D-quinovose. Finally, the chemical mechanism of AXS responsible for the biosynthesis of UDP-apiose has been investigated. In vitro activity assay using UDP-2F-glucuronic acid showed that the analog is a competitive inhibitor of AXS. A coupled assay strategy was also developed to investigate the chemical mechanism of AXS in the reverse direction. In addition, the stereospecificity of two separate hydride transfer steps of AXS reaction has been firmly established.