Dissertation
Allosteric regulation of pyruvate kinase muscle isoform 2
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2020
DOI: 10.17077/etd.005666
Abstract
Pyruvate kinase muscle isoform 2 is a key glycolytic enzyme, which is essential for the growth and proliferation of cancer cells and, consequently, it is tightly regulated in tumors by small molecules and posttranslational modifications (PTMs). Previous studies have identified amino acids (AAs), such as serine, alanine, phenylalanine, tryptophan, and posttranslational modifications, such as acetylation and phosphorylation, to be involved in allosteric regulation of PKM2. However, the molecular determinants and molecular mechanism involved in the enzymes regulation has not been understood well. Our biochemical, biophysical, and crystallographic studies of PKM2 with asparagine, aspartic acid, valine, and cysteine show that the non-polar side chains of valine and cysteine inhibit PKM2 whereas, the polar side chains of asparagine and aspartic acid activate PKM2 by altering the oligomeric state of the enzyme. Site-directed mutagenesis, coupled with activity assays and gel filtration studies, showed that a β strand between the active site and the AA binding pocket was responsible for bidirectional signaling between the sites. Also, our crystallographic studies of PTM mimetics of PKM2 showed that phosphorylation and acetylation of the enzyme cause minor structural changes in the binding pocket of fructose 1, 6 bisphosphate (FBP), which is an allosteric activator of PKM2. This leads to a decrease in the FBP binding affinity of the enzyme, which causes a shift in the oligomeric state of PKM2 from a tetramer to a dimer. Overall, our explanation of the allosteric regulation mechanism of PKM2 will aid in the development of small molecule agonists and antagonists against PKM2, which can influence the proliferation of tumors.
Details
- Title: Subtitle
- Allosteric regulation of pyruvate kinase muscle isoform 2
- Creators
- Suparno Nandi
- Contributors
- Mishtu Dey (Advisor)Edward G. Gillan (Advisor)Leonard R. MacGillivray (Committee Member)Daniel M. Quinn (Committee Member)David F. Wiemer (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Chemistry
- Date degree season
- Autumn 2020
- DOI
- 10.17077/etd.005666
- Publisher
- University of Iowa
- Number of pages
- xvi, 174 pages
- Copyright
- Copyright 2020 Suparno Nandi
- Language
- English
- Description illustrations
- illustrations (chiefly color)
- Description bibliographic
- Includes bibliographical references (pages 165-174).
- Public Abstract (ETD)
Pyruvate kinase muscle isoform 2 (PKM2) is a human enzyme, which helps in the metabolism of glucose and cancer gene expression in tumors. Glucose is essential for the cancer cells to survive, and the expression of cancer genes allows them to proliferate. The activity of the enzyme has been reported to be tightly regulated by several amino acids (AAs), as well as certain chemical reactions, such as acetylation and phosphorylation. However, the exact mechanism of PKM2 regulation in tumors is unknown. In the present study, using structural, biophysical, and biochemical studies, we have shown that water-soluble AAs activate the enzyme, while water-insoluble AAs inhibit the enzyme. The activation and inhibition occur by interchanging the enzyme between its native, active four-subunit, and inactive two-subunit structure. The activation mechanism by the water-soluble AAs is triggered by specific contacts between the AAs and the enzyme, which is lacking in the case of the water-insoluble AAs. We also showed that acetylation and phosphorylation of PKM2 break down the enzyme from a four-subunit to a two-subunit structure by reducing its binding affinity to fructose 1, 6 bisphosphate (FBP), which is an activator of PKM2. Our structural studies of PKM2 showed that subtle changes in parts of the FBP binding pocket, which are involved in binding FBP led to the decrease in the FBP binding affinity of the enzyme. In the future, our results can prove invaluable in the development of drugs against PKM2, which can help us in controlling the spread of tumors in the body.
- Academic Unit
- Chemistry
- Record Identifier
- 9984036790502771
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