enzyme kinetics and mechanisms ayesha amin, omkar baxi, laura gay, neha limaye, andrew massaro,...
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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
Enzyme and CatalysisP
oten
tial
En
ergy
Reaction Course
1. Transition State Stabilization
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)
Purine metabolism enzyme responsible for converting adenosine with water to inosine and ammonia.
http://sgc.utoronto.ca/SGC-WebPages/StructureDescription/2AMX.php
Adenosine Deaminase
ON
OHHO
N
N N
N
CH2OH
NH2 O
CH2OH
N
N N
NH
ON
OHHO
H2O
NH3
Adenosine Inosine
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
Key role in immunity and inflammation Adenosine with active stress and hypoxia
balances oxygen supply Stimulates angiogenesis
www.medem.com
ReactionsMichaelis- menten equation:
Model:
Michaelis constant:
Vmax= k2[ Et]
Lineweaver-Burk:y = mx + b
www.nsr.bioeng.washington.edu
Adenosine Deaminase
ON
OHHO
N
N N
N
CH2OH
NH2 O
CH2OH
N
N N
NH
ON
OHHO
H2O
NH3
Adenosine Inosine
Beer’s Law
Absorbance (λ)= C · l · ε λ
Absorbance: amount of light absorbed at a definite wavelengthC: concentrationl: 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
Experiment procedure1. Determine ε0 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 beer’s law 2. Find Vmax and Km
-Create 7 solutions using H20, HEPES, 10 uL of adenosine deaminase, and varying
adenosine concentrations
-run solutions at 264 nm and plot data
Monitoring rate of reactionKinet ic / Time: 15 um Adenosine
y = -0.0011x + 0.2268
R2 = 0.9773
0.222
0.223
0.224
0.225
0.226
0.227
0.228
0 1 2 3 4 5
Time (min)
Abs
Baseline Data
y = 3.09E+01x + 3.20E+06
R2 = 9.67E-01
0
2000000
4000000
6000000
8000000
10000000
12000000
14000000
16000000
0 100000 200000 300000 400000
1/ s0
1/
v0
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)
Adenosine and its Analogs
N
NN
N
NH2
O
OHOH
HH
HH
HO
N
NN
N
Cl
O
OHOH
HH
HH
HO
N
NN
N
NH2
O
OHOH
HH
HH
HO
N
NN
N
HN
O
OHOH
HH
HH
HOCl
Adenosine 6-Chloroadenosine
2-Chloroadenosine N6-Cyclohexyladenosine
6-Chloroadenosine
Kinetic/Time: 16um Chloro 10 minutes
y = -0.00023x + 0.15395
R2 = 0.97837
0.1515
0.152
0.1525
0.153
0.1535
0.154
0.1545
0 2 4 6 8 10 12
Time (min)
Abs
(mg/
mL)
AnalysisDouble Reciprocal Plot for 6-Chloroadenosine
y = 22.907x + 9E+06
R2 = 0.825
0
2000000
4000000
6000000
8000000
10000000
12000000
14000000
16000000
18000000
0 50000 100000 150000 200000 250000 300000 350000
1/[s]
1/[v
]
λ(nm) Abs ε (Abs/(M·cm))Vmax(M/s)
2.0x10-4 U/ml ADA
Km(µM)
Adenosine 264 0.315 6300 6.8x10-9 11.85
6-Chloroadenosine 269 0.105 2100 1.85x10-10 2.5
N6-CyclohexyladenosineTest for Inhibition with Cyclohexyladensine
0
0.0005
0.001
0.0015
0.002
0.0025
0.003
0.0035
0.004
0.0045
0.005
0 0.1 1 10 20 30
Concentration (um)
Reacti
on
Rate
(S
lop
e)
2-Chloroadenosine
0
0.0002
0.0004
0.0006
0.0008
0.001
0.0012
0.0014
5 10 20 30
Concentration 2-Chloroadenosine (uM)
Rate
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