Enzyme Kinetics and Mechanisms Ayesha Amin, Omkar Baxi,

Enzyme Kinetics and Mechanisms Ayesha Amin, Omkar Baxi,

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 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) 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 Potential Energy Enzyme and Catalysis 1. Transition State Stabilization Reaction Course 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 Adenosine Deaminase (ADA)

http://sgc.utoronto.ca/SGC-WebPages/ StructureDescription/2AMX.php Purine metabolism enzyme responsible for converting adenosine with water to inosine and ammonia. Adenosine Deaminase NH2 N CH2OH O N O N N CH2OH

O N N NH3 HO OH Adenosine NH H2O HO Inosine OH

N 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 ADA and Coronary Artery Disease www.medem.com Key role in immunity and inflammation Adenosine with active stress and hypoxia balances oxygen supply Stimulates angiogenesis www.nsr.bioeng.washington.e du Reactions

Michaelis- menten equation: Model: Michaelis constant: Vmax= k2[ Et] Lineweaver-Burk: y = mx + b Adenosine Deaminase NH2 N CH2OH O N O N N

CH2OH O N N NH3 HO OH Adenosine NH H2O HO Inosine OH

N Beers Law Absorbance ()= C l www.biocompare.co m Absorbance: amount of light absorbed at a definite wavelength C: concentration l: pathway (always one cm) : extinction coefficient: units = Abs mol. cm - absorbance of a given wavelength of light per mole of a compound Experiment procedure 1. Determine 0 for adenosine -

Find optimum concentration (50um) Run baseline of H20, buffer (hepes), and 50 um inosine Scan 50 um adenosine from 220 to 300nm Use peak in beers law 2. Find Vmax and Km -Create 7 solutions using H20, HEPES, 10 of adenosine deaminase, and varying adenosine concentrations -run solutions at 264 nm and plot data uL Monitoring rate of reaction Kinet ic / Time: 1 5 umAdeno s ine 0 .2 2 8 0 .2 2 7 Abs 0 .2 2 6 0 .2 2 5

0 .2 2 4 0 .2 2 3 y = -0 .0 0 1 1 x + 0 .2 2 6 8 2 R = 0 .9 7 7 3 0 .2 2 2 0 1 2 3 Time (min) 4 5 Baseline Data 16000000

14000000 12000000 1 / v0 10000000 8000000 6000000 4000000 y = 3 .0 9 E +0 1 x + 3 .2 0 E +0 6 2 R = 9 .6 7 E -0 1 2000000 0 0 100000 200000 1/ s0

300000 400000 Process Contd determine if the derivatives are direct inhibitors: same procedure as for adenosine Obtain optimal wavelengths and absorbencies and calculate 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) Adenosine and its Analogs NH2 N N Cl N

N N N HO N N HO O O H H OH

OH H H H H OH OH H H 6-Chloroadenosine Adenosine

NH2 N N HO HN N N N N Cl N N HO O

O H H OH OH H H 2-Chloroadenosine H H OH

OH H H N6-Cyclohexyladenosine 6-Chloroadenosine Kinetic/Time: 16um Chloro 10 minutes 0.1545 Abs (mg/mL) 0.154 0.1535 0.153 0.1525 y = -0.00023x + 0.15395 2 R = 0.97837 0.152

0.1515 0 2 4 6 Time (min) 8 10 12 Analysis Double Reciprocal Plot for 6-Chloroadenosine y = 22.907x + 9E+06 R2 = 0.825 18000000 16000000

14000000 1/[v] 12000000 10000000 8000000 6000000 4000000 2000000 0 0 50000 100000 150000 200000 250000

300000 350000 1/[s] (nm) Abs (Abs/(Mcm)) Vmax(M/s) 2.0x10-4 U/ml ADA Adenosine 264 0.315 6300

6.8x10-9 11.85 6-Chloroadenosine 269 0.105 2100 1.85x10-10 2.5 Km(M) N6-Cyclohexyladenosine Test for Inhibition with Cyclohexyladensine R e a c t io n R a t e ( S lo p e )

0.005 0.0045 0.004 0.0035 0.003 0.0025 0.002 0.0015 0.001 0.0005 0 0 0.1 1 10 Concentration (um) 20

30 2-Chloroadenosine 0.0014 0.0012 0.001 Rate 0.0008 0.0006 0.0004 0.0002 0 5 10 20 Concentration 2-Chloroadenosine (uM)

30 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. 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 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

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