Enzyme Kinetics and Mechanism Karen Cao Edward Lee

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/hhpajx 5. gif

What is an enzyme? • Catalyzes a chemical reaction by lowering activation energy • Affected by temperature and p. H • Essential within human body Chasin, Lawrence & Mowshowitz, Deborah. (2006, September). Lec. 6. Biol C 2005/F 2401 Columbia University. New York, NY. Retrieved on 8 August, 2007 from http: //www. columbia. edu/cu/biology/courses/c 2005/pu rves 6/figure 06 -14. jpg

Examples of Enzymes Enzyme Nonenzymatic t 1/2 knon (s-1) kcat (s-1) Rate enhancement (kcat/knon) OMP decarboxylase 78 000 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 1 VFL. png. (2007). Wikipedia. Retrieved August 1, 2007 from http: //en. wikipedia. org/wiki/Image: Adenosine_deaminase_1 VFL. png

Adenosine • Critical nucleoside • Backbone of various biological structures ØAdenosine triphosphate (ATP) ØCellular receptors (G-proteins) • Prevents tissue damage during hypoxia, ischemia, and seizure activity

Adenosine to Inosine NH 2 N CH 2 OH O N N NH 3 HO OH Adenosine NH H 2 O HO Inosine OH N

ADA Complications • Severe Combined Immunodeficiency Syndrome (SCIDS) • Lymphoma • Hemolytic Anemia “A T Cell killing a cancer cell. ” (2007). Tcells. 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/honors 2000/group 08/ images/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 • p. H dependence of two ? substrates ØAdenosine Ø 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.

Adenosine 6 -Chloroadenosine NH 2 N N HO Cl N N HO O H H H H OH OH Adenosine H H H OH OH 6 -Chloroadenosine N N

Overview of experiment • Determine rates of reaction of both adenosine and 6 -chloroadenosine at varying concentrations and p. Hs. • Calculate and compare rate constants to establish which reaction is more sensitive to p. H

Why This Works • At higher p. Hs, the solution will neutralize the acidic side chain before it can protonate the NH 2 • 6 -Chloroadenosine does not need a proton to continue with reaction • Therefore, 6 -Chloroadenosine will be less dependent on p. H and show higher reaction rates. The Science Company. (2007). Toward Understanding p. H. Retrieved 7 August, 2007 from www. sciencecompany. com/iages/phscale. gif

Projected k 2 Graphs Case 1: p. H dependence comes from the protonation of the amine group Adenosine Case 2: p. H dependence comes from the protonation of the 1 N or denaturation of the protein 6 -Chloroadenosine

Materials • • Adenosine solution Adenosine deaminase 6 -Chloroadenosine Buffers of varying p. H 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 p. H • 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

Michaelis-Menten Kinetics Berg, J. M. , Tymoczko, J. L. , & Stryer, L. (2007. ) Biochemistry. (6 th ed. ) New York: W. H. Freeman and Company.

Double Reciprocal Plot y = m • x + b Berg, J. M. , Tymoczko, J. L. , & Stryer, L. (2007. ) Biochemistry. (6 th ed. ) New York: W. H. Freeman and Company.

p. H k 2(Ad) p. H k 2(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

Conclusions § 2 preliminary conclusions ØAdenosine is more p. H sensitive than 6 chloroadenosine ØImportance of acidic side chains and protonation of amine group in p. H dependence

Further Testing • Determinations of more k₂ values ØTest adenosine and 6 -Cl-adenosine at more p. H’s and more concentrations • Testing of individual amino acid groups within enzyme ØReplacement of amino acid groups via mutagenesis

Applications • Comprehension of underlying ADA catalysis mechanism allows for more effective ADA inhibitors • Major medical implications ØSCIDS ØLymphomas ØMetabolic 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 ØT 1 ballers@gmail. com

Sources 1. 2. 3. 4. 5. 6. Campbell, N. A. & Reece J. B. (2005). Biology. 123 Adenosine Deaminase 1 VFL. png. (2007). Wikipedia. Retrieved August 1, 2007 from http: //en. wikipedia. org/wiki/Image: Adenosine_deaminase_1 VFL. 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/honors 2000/group 08/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. (6 th ed. ) New York: W. H. Freeman and Company.

Sources • Catalysis. http: //www. columbia. edu/cu/biology/courses/c 2005/purves 6/figure 06 -14. jpg • http: //upload. wikimedia. org/wikipedia/commons/thumb/c/c 6/Adenosine_deaminase_1 VFL. pn g/593 px-Adenosine_deaminase_1 VFL. png • Slide 1 http: //biomechanics. ecs. umass. edu/HHPAJX/hhpajx 5. gif