Loading...

Cloning, Expression and Purification of Yeast Fumarase Enzyme, in Order to Find Small Organic Molecules as its Allosteric Modulators

Navaee Lavasani, Elahe | 2024

0 Viewed
  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 57106 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Kalhor, Hamid Reza
  7. Abstract:
  8. Enzymes are biological catalysts that carry out chemical reactions in a stereoselective manner. Fumarate hydratase (abbreviated as fumarase) is a metabolic enzyme found in both mitochondrial and cytosolic isoforms. Its mitochondrial form functions in the Krebs cycle (TCA) and specifically converts fumarate into L-malate. In recent studies, the role of the cytosolic form of this enzyme in the repair of DNA double-strand breaks has been elucidated. Fumarase and its precursors are indirectly related to many metabolic pathways, including phenylalanine and tyrosine metabolism, urea cycle, etc. As a result, it is very important to know the factors affecting this enzyme and its activity. In this research, at first, the yeast fumarase was cloned by using PCR based methodology and restriction enzymes. It then was expressed and purified in an optimized condition. The kinetic parameters of yeast fumarase was measured at ambient temperature in the presence of L-malate and fumarate precursors. The enzyme efficiency in the presence of L-malate and fumarate was 3.46 x 104 (1/s.mM) and 2.20 x 105 (1/s.mM), respectively. The effect of small organic molecules on the fumarase activity was investigated by inspiration from the cycles associated with it, in order to find its allosteric modulators. Among these molecules were L-arginine and L-tyrosine, uric acid and vitamin B5. They did not have a significant effect on the enzyme activity. Using computational approach, a library of 5387 organic compounds was selected to be docked on the 3D structure of fumarase to select the best cavity and structure in terms of binding affinity and conformation. The two top selected molecules had Dioxan-4,6-dione and prymidine-2,4,6-trione rings
  9. Keywords:
  10. DNA Double Strand ; Molecular Docking ; Fumarase Enzyme ; Krebs Cycle ; Allosteric Effects ; Enzyme Kinetics ; Cloning

 Digital Object List

 Bookmark

No TOC