Malonyl-thioesters are reactive at the thioester
carbonyl and the carboxylate moieties, as seen in acyl transfer or hydrolysis
and decarboxylation. Enzymes use these reactive centers to perform different
enzyme chemistry throughout metabolism. This enzyme chemistry coupled with the
inherent reactivity of malonyl-thioesters makes structure-function studies
difficult. When malonyl-thioesters are used for structure-function studies, it
usually results in a hydrolyzed or decarboxylated product. There are examples,
however, where this is overcome, many of which are discussed throughout this
thesis. To overcome the inherent reactivity of malonyl-thioesters and enzymes,
analogs have been synthesized to perform structure-function studies. Initial
studies focused on altering the thioester carbonyl to limit hydrolysis and
decarboxylation; however, these studies revealed the importance of retaining
the thioester carbonyl to be positioned in the oxyanion hole. My thesis work
focused on the synthesis, characterization, and use in structure-function
studies of malonyl-thioester analogs that either preserve the thioester
carbonyl or alter it to an ester or amide, and alter the carboxylate to a
sulfonate or nitro group. After synthesizing the methylmalonyl-CoA analogs, we
performed structure-function studies with methylmalonyl-CoA decarboxylase. This
case study revealed the potential of these analogs to both inhibit
decarboxylase activity and their use in structure-function studies to gain
mechanistic insights. This successful study prompted us to continue these
structure-function studies in enzymes with different chemistries such as an
epimerase or bi-functional acyltransferase/decarboxylase. The widespread use of
these methylmalonyl-CoA analogs also motivated us to add more malonyl-thioester
analogs to our toolbox. I have preliminary data that these malonyl-thioester
analogs inhibit β-keto-acyl-synthase III, an enzyme involved in fatty acid
production in <i>E. coli</i>. This
inhibition gives us confidence that these analogs will be useful in
structure-function studies that will reveal answers to long standing mechanism
and protein-protein interaction questions in the polyketide and fatty acid
synthase field.
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/11324174 |
| Date | 05 December 2019 |
| Creators | Lee M Stunkard (8086712) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/UNVEILING_ENZYMATIC_MECHANISMS_WITH_MALONYL-THIOESTER_ISOSTERES/11324174 |
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