| |
| author | Julie Mary Alex |
| title |
Ca2+ Catalyzed Adenosine 3', 5'-Cyclic Monophosphate Hydrolysis: Insights into the Role of Metals in Phosphodiesterase Active Sites
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| abstract |
Cyclic adenosine monophosphate (cAMP) is an important secondary messenger found within biological systems. A family of phosphodiesterases (PDEs) hydrolyze
cAMP into 5' adenosine monophosphate which is further processed into adenosine to alter signaling pathways. PDEs have been a pharmaceutical target for
diseases, including end-stage heart failure, autoimmune diseases/allergies, and contractile dysfunction, that are impacted by extreme cAMP fluxes. A new
model system using calcium (Ca2+) was analyzed for the production of 3'AMP and 5'AMP along with the mechanism of metal ion catalysis. In this
study, kinetic assays with high pressure liquid chromatography were used with Ca2+ ions and cAMP to collect rate constants, and to conduct
product and reactant analysis. The products were confirmed to be 3'AMP and 5'AMP using electrospray ionisation time-of-flight (ESI-TOF) mass spectroscopy
and comparison with HPLC standards. The current model system shows an increase in the ratio of 3'AMP versus 5'AMP production as [Ca2+] increase.
Once rate constants were collected, they were compared with the current DNA and RNA models. Graphing the rate constants versus Ca2+ concentration
shows a second order curve fit similar to the DNA model, p-nitrophenyl-thymidine- 5'-phosphate (T5PNP), and different to the RNA model. The k0
with no Ca2+ is 2.6*10-4 s-1, k1st with 1st order Ca2+ is 1.6*10-2
M-1s-1, and k2nd with 2nd order Ca2+ is 2.3*10-1 M-2s-1 for the
pseudo-first order reaction in regards to Ca2+. When analyzing the difference in catalysis of NaOH and Ca2+, the ΔΔG☨,
difference in activation energy stabilization, is 18 kJ/mol. Isotope reactions with alkaline phosphatase indicate that the hydroxide attacks the phosphorus
instead of the carbon. This model system allows for intermediate analysis to determine a dual ion mechanism and attack at the phosphorus.
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| school | The College of Liberal Arts, Drew University |
| degree | B.A. (2018) |
| advisor | Adam Cassano |
| committee | Joanna Miller
Sandra Keyser |
| full text | JMAlex.pdf |
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