Enzyme Enzyme Kinetics Kinetics and Mechanism 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.ecs.uma ss.edu/HHPAJX/ hhpajx5.gif
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EnzymeKinetics and Mechanism Enzyme Kinetics and Mechanism Karen Cao, Edward Lee, Jennifer Liu, Dea Yong Park, Sharmila Railkar, Jyotsna Ramachanadran,
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EnzymeEnzyme Kinetics Kinetics and Mechanismand Mechanism
Karen Cao, Edward Lee, Jennifer Liu, Dea Yong Park, Sharmila
Railkar, Jyotsna Ramachanadran, Jason Stickel, Laura Tiedemann,
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/purves6/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
• Prevents tissue damage during hypoxia, ischemia, and seizure activity
Adenosine to Inosine
ON
OHHO
N
N N
N
CH2OH
NH2 O
CH2OH
N
N N
NH
ON
OHHO
H2O
NH3
Adenosine Inosine
ADA Complications• Severe Combined
Immunodeficiency Syndrome (SCIDS)
• Lymphoma• Hemolytic Anemia
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/babybubble.jpg
“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
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 substratesAdenosine6-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.
gselee
Is hisitidine the correct basic amino acid?
N
NN
N
NH2
O
OHOH
HH
HH
HO
N
NN
N
Cl
O
OHOH
HH
HH
HO
Adenosine 6-Chloroadenosine
Adenosine 6-Chloroadenosine
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
Case 2: pH dependence comes from the protonation of the 1N or denaturation of the protein
6-ChloroadenosineAdenosine
Materials• Adenosine solution• Adenosine deaminase• 6-Chloroadenosine• Buffers of varying pH
• Distilled Water• Micropipettes• Microcentrifuge
tubes• Spectrophotometer
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
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
gskweibert
Absorbance vs. Time of 60 microM 6-Chloroadenosine at pH 8.9 y = -5.12E-05x + 4.38E-01
R2 = 9.92E-01
0.4050.41
0.4150.42
0.4250.43
0.4350.44
0 100 200 300 400 500 600 700
Time (sec.)
Ab
so
rba
nc
e
Absorbance vs. Time of 50 microM Adenosine at pH 8.9
Sources1. Campbell, N. A. & Reece J. B. (2005). Biology. 1232. Adenosine Deaminase 1VFL.png. (2007). Wikipedia. Retrieved
August 1, 2007 from http://en.wikipedia.org/wiki/Image:Adenosine_deaminase_1VFL.png
3. “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
4. 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/babybubble.jpg
5. 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. Berg, J. M., Tymoczko, J. L., & Stryer, L. (2007.) Biochemistry. (6th ed.) New York: W.H. Freeman and Company.