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Enzyme Kinetics and
Mechanism
Karen Cao, Edward Lee, Jennifer Liu,
Dea Yong Park, Sharmila Railkar,
Jyotsna Ramachanadran, Jason Stickel,
Laura Tiedemann, Lindsay Vendetta,
Kurt Weiberth, Caitlin Williams
•http://biomechanics.e
cs.umass.edu/HHPAJ
X/hhpajx5.gif
What is an enzyme?
• Catalyzes a
chemical reaction by
lowering activation
energy
• Affected by
temperature and pH
• Essential within
human body
Chasin, Lawrence & Mowshowitz, Deborah. (2006,
September). Lec. 6. Biol C2005/F2401 Columbia
University. New York, NY. Retrieved on 8 August,
2007 from
http://www.columbia.edu/cu/biology/courses/c2005/pu
rves6/figure06-14.jpg
Examples of Enzymes
Enzyme
Nonenzymatic t1/2
knon (s-1)
kcat
(s-1)
Rate
enhancement
(kcat/knon)
OMP
decarboxylase
78 000 000 years
2.8 x 10-16
39
1.4 x 1017
Staphylococcal
nuclease
130 000 years
1.7 x 10-13
95
5.6 x 1014
Adenosine
deaminase
120 years
1.8 x 10-10
370
2.1 x 1012
AMP
nucleosidase
69 000 years
1.0 x 10-11
60
6.0 x 1012
Cytidine
deaminase
69 years
3.2 x 10-10
299
1.2 x 1012
CITATION
Enzymes
• Active Site- the specific
portion of an enzyme
that attaches to the
substrate
• Substrate- the reactant
on which an enzyme
works
Campbell, N. A. & Reece
J. B. (2005). Biology.
pp. 123
Adenosine Deaminase (ADA)
• T cell development
• Neurotransmission
• Blood flow
• Platelet aggregation
• Regulates adenosine
levels
Adenosine Deaminase 1VFL.png. (2007). Wikipedia. Retrieved
August 1, 2007 from
http://en.wikipedia.org/wiki/Image:Adenosine_deaminase_1VFL
.png
Adenosine
• Critical nucleoside
• Backbone of various biological structures
Adenosine triphosphate (ATP)
Cellular receptors (G-proteins)
• Prevents tissue damage during hypoxia,
ischemia, and seizure activity
Adenosine to Inosine
NH2
N
CH2OH
O
N
O
N
N
CH2OH
O
N
N
NH3
HO
OH
Adenosine
NH
H 2O
HO
Inosine
OH
N
ADA Complications
• Severe Combined
Immunodeficiency
Syndrome (SCIDS)
• Lymphoma
• Hemolytic Anemia
“A T Cell killing a cancer cell.” (2007). T-cells.
Retrieved August 3, 2007 from
http://www.sciencemuseum.org.uk/online/lifecycle/116.asp
Cavazzana-Calvo, M. & Hacein-Bey, S. Gene Therapy:
Bursting the Bubble of SCIDS. (2001). University of
Arizone. Retrieved August 1, 2007 from
http://student.biology.arizona.edu/honors2000/group08/i
mages/babybubble.jpg
How ADA catalyzes
Wilson, D. K. et. al. Atomic Structure of
Adenosine Deaminase Complexed with a
Transition-State Analog: Understanding
Catalysis and Immunodeficiency Mutations.
(1991.) Science 252 (5010). 1278.
How ADA catalyzes
Wilson, D. K. et. al. Atomic Structure of
Adenosine Deaminase Complexed with a
Transition-State Analog: Understanding
Catalysis and Immunodeficiency Mutations.
(1991.) Science 252 (5010). 1278.
How ADA catalyzes
Wilson, D. K. et. al. Atomic Structure of
Adenosine Deaminase Complexed with a
Transition-State Analog: Understanding
Catalysis and Immunodeficiency Mutations.
(1991.) Science 252 (5010). 1278.
How ADA catalyzes
Wilson, D. K. et. al. Atomic Structure of
Adenosine Deaminase Complexed with a
Transition-State Analog: Understanding
Catalysis and Immunodeficiency Mutations.
(1991.) Science 252 (5010). 1278.
How ADA catalyzes
Wilson, D. K. et. al. Atomic Structure of
Adenosine Deaminase Complexed with a
Transition-State Analog: Understanding
Catalysis and Immunodeficiency Mutations.
(1991.) Science 252 (5010). 1278.
Purpose
• Begin attempts to identify the
functional group which
protonates the amine leaving
group
• pH dependence of two
?
substrates
Adenosine
6-Chloroadenosine
Wilson, D. K. et. al. Atomic Structure of Adenosine Deaminase
Complexed with a Transition-State Analog: Understanding
Catalysis and Immunodeficiency Mutations. (1991.) Science
252 (5010). 1278.
6-Chloroadenosine
Adenosine
NH 2
Cl
N
N
N
N
HO
N
N
N
HO
O
H
O
H
H
H
H
OH
OH
Adenosine
H
H
H
OH
OH
6-Chloroadenosine
N
Overview of experiment
• Determine rates of reaction of both
adenosine and 6-chloroadenosine at
varying concentrations and pHs.
• Calculate and compare rate constants to
establish which reaction is more sensitive
to pH
Why This Works
• At higher pHs, the solution will
neutralize the acidic side chain
before it can protonate the NH2
• 6-Chloroadenosine does not
need a proton to continue with
reaction
• Therefore, 6-Chloroadenosine
will be less dependent on pH
and show higher reaction rates.
The Science Company. (2007). Toward Understanding
pH. Retrieved 7 August, 2007 from
www.sciencecompany.com/iages/phscale.gif
Projected k2 Graphs
Case 1: pH dependence comes
from the protonation of the amine
group
Adenosine
Case 2: pH dependence comes
from the protonation of the 1N or
denaturation of the protein
6-Chloroadenosine
Materials
•
•
•
•
Adenosine solution
Adenosine deaminase
6-Chloroadenosine
Buffers of varying pH
Micropipette. (2007). Biokits.com. Retrieved 8
August, 2007 from http://www.biokits.com
DU® 530 Life Science UV/Visible
Spectrophotometers. (2007). Retrieved 8
August from http://www.biocompare.com
•
•
•
•
Distilled Water
Micropipettes
Microcentrifuge tubes
Spectrophotometer
Procedure
• Use varying concentrations of adenosine solution
and 6-chloroadenosine solution at each pH
• Add adenosine deaminase
• Run sample through spectrophotometer for
duration of reaction to analyze rate of reaction
– Beer’s Law: Abs.  concentration
• Compress data and compare reaction rates of
adenosine deaminase and 6-chloroadenosine
y = -5.75E-04x + 7.01E-01
Absorbance
Absorbance vs. Time of 50 microM
Adenosine at pH 8.9
2
R = 1.00E+00
0.7100
0.7000
0.6900
0.6800
0.6700
0.6600
0.6500
0.6400
0.6300
0.6200
0.6100
0
20
40
60
80
100
120
140
160
Time (sec.)
Absorbance
Absorbance vs. Time of 60 microM
6-Chloroadenosine at pH 8.9
y = -5.12E-05x + 4.38E-01
R2 = 9.92E-01
0.44
0.435
0.43
0.425
0.42
0.415
0.41
0.405
0
100
200
300
400
Time (sec.)
500
600
700
Michaelis-Menten Kinetics
vmax [ s]
vo 
k m  [ s]
vmax  k2  [ E ]t
Berg, J. M., Tymoczko, J.
L., & Stryer, L. (2007.)
Biochemistry. (6th ed.)
New York: W.H. Freeman
and Company.
k 1  k 2
KM 
k1
M ichae lis-M e nte n Chart for
Ade nosine at pH 8.9
0.00000008
0.00000007
0.00000006
Vo
0.00000005
0.00000004
0.00000003
0.00000002
0.00000001
10.00
0
[S]
M ichae lis-M e nte n Chart for
6-Chloroade nosine at pH 8.9
2.00E-08
1.80E-08
1.60E-08
1.40E-08
Vo
1.20E-08
1.00E-08
8.00E-09
6.00E-09
4.00E-09
2.00E-09
0.00E+00
0
20
40
60
[S]
80
100
120
Double Reciprocal Plot
1 KM
1
1



vo Vmax [ S ] Vmax
y = m • x + b
Berg, J. M., Tymoczko, J. L., &
Stryer, L. (2007.) Biochemistry.
(6th ed.) New York: W.H.
Freeman and Company.
Double Reciprocal Chart for
Adenosine at pH 8.9
y = 4.58E+08x + 3.11E+06
R2 = 9.88E-01
60000000
50000000
1/Vo
40000000
30000000
20000000
10000000
0
0
0.02
0.04
0.06
0.08
0.1
0.12
1/[S]
Double Reciprocal Chart for
6-Chloroadenosine at pH 8.9
y = 3.64E+09x + 3.11E+07
R2 = 9.84E-01
800000000
700000000
600000000
1/Vo
500000000
400000000
300000000
200000000
100000000
0
0.00
0.05
0.10
0.15
1/[S]
0.20
0.25
pH
k2(Ad)
pH
k2(Cl-Ad)
7.3
68.3
7.3
0.26
8.4
30.6
8.4
0.319
8.9
4.62
8.9
0.77
9.4
1.63
9.4
<0.08
80
0.8
60
0.6
40
0.4
20
0.2
0
0
7
7.5
8
Adenosine
8.5
pH
9
9.5
6-chloroadenosine
10
6-C l-Adenosine k2
Adenosine k2
pH vs. k2 for Adenosine and
6-Cl-Adenosine
Conclusions
 2 preliminary conclusions
Adenosine is more pH sensitive than 6chloroadenosine
Importance of acidic side chains and
protonation of amine group in pH
dependence
Further Testing
• Determinations of more k₂ values
Test adenosine and 6-Cl-adenosine at more
pH’s and more concentrations
• Testing of individual amino acid groups
within enzyme
Replacement of amino acid groups via
mutagenesis
Applications
• Comprehension of underlying ADA
catalysis mechanism allows for more
effective ADA inhibitors
• Major medical implications
SCIDS
Lymphomas
Metabolic disorders
Thank You
• Prudential and other sponsors
• Dr. Miyamoto
• Dr. Steven Surace
• Dr. Paul Victor Quinn, Sr.
• Myrna Papier
• Dr. Adam Cassano
• Jen Cowell
Questions? Comments?
Concerns?
•Email us at
[email protected]
Sources
1.
2.
3.
4.
5.
6.
Campbell, N. A. & Reece J. B. (2005). Biology. 123
Adenosine Deaminase 1VFL.png. (2007). Wikipedia. Retrieved August 1,
2007 from
http://en.wikipedia.org/wiki/Image:Adenosine_deaminase_1VFL.png
“A T Cell killing a cancer cell.” (2007). T-cells. Retrieved August 3, 2007
from http://www.sciencemuseum.org.uk/on-line/lifecycle/116.asp
Cavazzana-Calvo, M. & Hacein-Bey, S. Gene Therapy: Bursting the Bubble of
SCIDS. (2001). University of Arizone. Retrieved August 1, 2007 from
http://student.biology.arizona.edu/honors2000/group08/images/babybub
ble.jpg
Wilson, D. K. et. al. Atomic Structure of Adenosine Deaminase
Complexed with a Transition-State Analog: Understanding Catalysis
and Immunodeficiency Mutations. (1991.) Science 252 (5010). 1278.
Berg, J. M., Tymoczko, J. L., & Stryer, L. (2007.) Biochemistry. (6th ed.)
New York: W.H. Freeman and Company.
Sources
• Catalysis.
http://www.columbia.edu/cu/biology/courses/c2005/purves6/figure06-14.jpg
•
http://upload.wikimedia.org/wikipedia/commons/thumb/c/c6/Adenosine_deaminase_1VFL.pn
g/593px-Adenosine_deaminase_1VFL.png
•
Slide 1 http://biomechanics.ecs.umass.edu/HHPAJX/hhpajx5.gif