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.ecs.umass.edu/HHPAJX/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/purves6/figure06-14.jpg

Examples of Enzymes CITATION Enzyme Nonenzymatic t1/2 knon (s-1) kcat 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 Active Sites lower activation energy by: Acting as a template for substrate orientation Stressing the substrates and stabilizing the transition state Providing a favorable microenvironment Participating directly in the catalytic reaction 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 O N H C 2 H2O NH3 Adenosine Inosine

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/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/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 N H 2 O C l A d e n o s i 6 - h r a

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 Label (colors and axes) Adenosine 6-Chloroadenosine

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

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

pH k2(Ad) k2(Cl-Ad) 7.3 68.3 0.26 8.4 30.6 0.319 8.9 4.62 0.77 9.4 1.63 <0.08 Vmax=1/y-int Km=slope/y-int K2=

Conclusions 2 preliminary conclusions Adenosine is more pH sensitive than 6-chloroadenosine 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 T1ballers@gmail.com

Sources 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/babybubble.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. Ca

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.png/593px-Adenosine_deaminase_1VFL.png Slide 1 http://biomechanics.ecs.umass.edu/HHPAJX/hhpajx5.gif