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| Title: | Comparative Properties in Kinetics and Inhibition of Human and Drosophila Lysyl Oxidase-Like Enzymes |
| Authors: | Culpepper, Matthew Brice |
| Advisors: | Givens, Richard S. |
| Commitee Members: | Schowen, Richard L.
Hanson, Paul R. |
| Keywords: | Chemistry, Biochemistry
Enzyme
Inhibition
Kinetics
Lysyl oxidase-like
Model compound |
| Issue Date: | 28-Jul-2009 |
| Publisher: | University of Kansas |
| Extent: | 107 pages |
| Type: | Thesis |
| Degree Level: | M.S. |
| Discipline: | Chemistry |
| Abstract: | Lysyl oxidase-like enzymes are found in an array of organisms and have been linked to a variety of diseases. The research described in this thesis focuses on the characterization and analysis of lysyl oxidase-like proteins found in Drosophila Melanogaster and one in Homo sapiens. The long-term goal is to fully characterize the enzymes which could lead to developing specific inhibitors that can be used to prevent certain diseases.
Recombinant forms of lysyl oxidase-like (LOXL) namely the D. melanogaster and H. sapiens enzyme forms are being investigated. Expressions of the recombinant forms of the enzymes were incorporated into a eukaryotic expression system and contain a Strep-tag II sequence for purification. D. melanogaster Schneider 2 (S2) cells were chosen due to inclusion bodies obtained using an E. coli expression system. The S2 expression system produced soluble forms of both enzymes; however the DLOXL1 form showed no substrate activity. This lack of activity was linked to the active site cofactor lysine tyrosylquinone (LTQ). To facilitate the formation of LTQ, all metal was removed and the natural metal Cu2+ was reintroduced which produced active protein.
Kinetic parameters kcat, Km, and kcat/Km were determined using 10 possible substrate amines. Substrate inhibition was observed with tyramine and dopamine with DLOXL1. Substrate inhibition was also observed with tyramine and octopamine for HLOXL2. Out of the 10 substrates serotonin and benzylamine demonstrated no detectable activity for both enzymes. However, tropoelastin and lysine showed no detectable activity with HLOXL2. The lack of activity for tropoelastin is a contradicting result to the purposed roles of LOX and LOXL enzymes. From the kinetic parameters a hypothesis can be made in regards to the substrates catalytic efficiency. The highest catalytic efficiency and turnover were related to substrates that contained a minimum of 2 non-branched carbons between the reactive amine and bulky functional groups.
Logarithmic plots of the kcat and kcat/Km values for DLOXL1 were plotted versus the log of kcat and kcat/Km values for HLOXL2. The results suggest that DLOXL1 and HLOXL2 share no common substrate similarities. This could suggest that DLOXL1 and HLOXL2 do not share similar functions or physiological roles.
Inhibition of DLOXL1 and HLOXL2 was examined using the known LOX inhibitor β-aminopropionitrile (βAPN). IC50 values of 3.9 and 6.5 μM with DLOXL1 and HLOXL2 respectively. Time-dependent studies were performed using the inhibitor βAPN incubated with HLOXL2 at various times. The results were plotted and shown in Chapter 3. Inhibition mechanisms were studied by using three βAPN concentrations and varying the substrate cadaverine. Graphs shown in Chapter 3 indicate that βAPN is a competitive inhibitor of HLOXL2.
A model compound was synthesized to mimic the LTQ cofactor. Confirmation of the model compound was performed by high-resolution mass spectrometry and UV-visible spectrometry. Formation of the quinone from the reduced form was monitored at 494 nm over time, which is shown in Chapter 4.
The concluding remarks address the overall information gathered and future studies that need to be performed in order to gain more information on this poorly understood enzyme which could ultimately lead to development of a selective inhibitor for lysyl oxidase-like proteins. |
| URI: | http://hdl.handle.net/1808/5587 |
| Appears in Collections: | Theses
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