1. Enzyme Kinetics and Mechanisms Ayesha Amin, Omkar Baxi, Laura Gay, Neha Limaye, Andrew Massaro, Daniel Nachajon, Albert Ng, Melanie Pastuck, Tara Weigand, and Rose Yu Dr. Adam Cassano and Jen Cowell

2. Thesis/Purpose  To determine how modifications to adenosine affect binding to the adenosine deaminase active site  Long term goal: developing inhibitors to adenosine deaminase (drugs to treat diseases)

3. What is an enzyme?  catalyzes a chemical reaction  Unique tertiary structure – active site binds to specific substrates  rate affected by temperature, pH, and concentrations of both enzyme and substrate. www.oak.cats.ohiou.edu

4. Enzyme and Catalysis Potential Energy Reaction Course 1. Transition State Stabilization

5. Inhibition of Enzymes  Inhibitors: molecules that bind to enzyme and slow down reaction  Competitive Inhibitors – bind at same active site as the substrate  Noncompetitive Inhibitors – bind at a different site Changes enzyme shape, altering active site www.eccentrix.com

6. Adenosine Deaminase (ADA)  Purine metabolism enzyme responsible for converting adenosine with water to inosine and ammonia. http://sgc.utoronto.ca/SGC- WebPages/StructureDescription/2AMX.php

7. Adenosine Deaminase O N OHHO N N N N CH 2 OH N H 2 O CH 2 OH N N N NH O N OHHO H2OH2O NH3NH3 AdenosineInosine

8. Did you know?  ADA imbalance within the body can result in a variety of health problems  High Levels of ADA activity are present in certain leukemias  Inhibition of ADA can stop growth of some cancerous cells

9. ADA and Coronary Artery Disease  Key role in immunity and inflammation  Adenosine with active stress and hypoxia balances oxygen supply  Stimulates angiogenesis www.medem.com

10. Reactions  Michaelis- menten equation:  Model:  Michaelis con stant :  V max = k 2 [ E t ]  Lineweaver-Burk: y = mx + b www.nsr.bioeng.washington.edu

12. Adenosine Deaminase O N OHHO N N N N CH 2 OH N H 2 O CH 2 OH N N N NH O N OHHO H2OH2O NH3NH3 AdenosineInosine

13. Beer’s Law Absorbance ( λ)= C · l · ε λ Absorbance: amount of light absorbed at a definite wavelength C: concentration l : pathway (always one cm) ε λ : extinction coefficient: units = Abs mol. · cm - absorbance of a given wavelength of light per mole of a compound www.biocompare.com

14. Experiment procedure 1.Determine ε 0 for adenosine -Find optimum concentration (50um) -Run baseline of H 2 0, buffer (hepes), and 50 um inosine -Scan 50 um adenosine from 220 to 300nm -Use peak in beer’s law 2. Find V max and K m -Create 7 solutions using H 2 0, HEPES, 10 uL of adenosine deaminase, and varying adenosine concentrations -run solutions at 264 nm and plot data

15. Monitoring rate of reaction

16. Baseline Data

17. Process Cont’d  determine if the derivatives are direct inhibitors: same procedure as for adenosine  Obtain optimal wavelengths and absorbencies and calculate ε for the 3 compounds  Compare reactivity with the enzyme for each (test for binding)  Create mixture of compound and adenosine, compare reactivity with original adenosine scan (Test for inhibition)

18. Adenosine and its Analogs

19. 6-Chloroadenosine

20. Analysis λ (nm) Abs ε (Abs/(M·cm)) V max (M/s) 2.0x10 -4 U/ml ADA K m (µM) Adenosine2640.31563006.8x10 -9 11.85 6-Chloroadenosine2690.10521001.85x10 -10 2.5

21. N6-Cyclohexyladenosine

22. 2-Chloroadenosine

23. Conclusion Of the two positions we examined, the 2 position did not show evidence of binding, and the 6 position showed possibility of binding, depending on the substituent.

24. Future Goals…  Repeat/verify our results More trials to determine uncertainty in our values different methods [liquid chromatography] to compare results  Different modifications [n6 position]  Varying environmental conditions pH Temperature

25. The End Thank you:  Laura and John Overdeck  Other Sponsors of NJGSS 2006  Director Miyamoto  Surace  Paul Quinn  Myrna Papier  Team Project Leader Dr. Cassano  Team Project Assistant Jen Cowell