EnzymeEnzyme Kinetics Kinetics and Mechanismand 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

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

structuresAdenosine triphosphate (ATP)Cellular receptors (G-proteins)

• 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.

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

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

y = -5.75E-04x + 7.01E-01

R2 = 1.00E+00

0.61000.62000.63000.64000.65000.66000.67000.68000.69000.70000.7100

0 20 40 60 80 100 120 140 160

Time (sec.)

Ab

so

rba

nc

e

Michaelis-Menten Kinetics

tEkv ][2max

1

21

k

kkKM

][

][max

sk

svv

mo

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

Michaelis-Menten Chart for 6-Chloroadenosine at pH 8.9

0.00E+00

2.00E-09

4.00E-09

6.00E-09

8.00E-09

1.00E-08

1.20E-08

1.40E-08

1.60E-08

1.80E-08

2.00E-08

0 20 40 60 80 100 120

[S]

Vo

Michaelis-Menten Chart for Adenosine at pH 8.9

0

0.00000001

0.00000002

0.00000003

0.00000004

0.00000005

0.00000006

0.00000007

0.00000008

10

.00

[S]

Vo

Double Reciprocal Plot

maxmax

1

][

11

VSV

K

vM

o

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 forAdenosine at pH 8.9

y = 4.58E+08x + 3.11E+06R2 = 9.88E-01

0

10000000

20000000

30000000

40000000

50000000

60000000

0 0.02 0.04 0.06 0.08 0.1 0.12

1/[S]

1/V

o

Double Reciprocal Chart for6-Chloroadenosine at pH 8.9

y = 3.64E+09x + 3.11E+07

R2 = 9.84E-01

0

100000000

200000000

300000000

400000000

500000000

600000000

700000000

800000000

0.00 0.05 0.10 0.15 0.20 0.25

1/[S]

1/V

o

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

pH vs. k2 for Adenosine and

6-Cl-Adenosine

0

20

40

60

80

7 7.5 8 8.5 9 9.5 10pH

Adenosi

ne k

2

0

0.2

0.4

0.6

0.8

6-C

l-A

denosi

ne k

2

Adenosine 6-chloroadenosine

Conclusions

2 preliminary conclusionsAdenosine is more pH sensitive than 6-

chloroadenosineImportance of acidic side chains and

protonation of amine group in pH dependence

Further Testing

• Determinations of more k₂ valuesTest adenosine and 6-Cl-adenosine at

more pH’s and more concentrations

• Testing of individual amino acid groups within enzymeReplacement of amino acid groups via

mutagenesis

Applications

• Comprehension of underlying ADA catalysis mechanism allows for more effective ADA inhibitors

• Major medical implicationsSCIDSLymphomasMetabolic 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

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.

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