Enzyme Kinetics and Mechanisms

Ayesha Amin, Omkar Baxi, Laura Gay, Neha Limaye, Andrew Massaro, Daniel Nachajon, Albert Ng, Melanie Pastuck,

Tara Weigand, and Rose Yu

Dr. Adam Cassano and Jen Cowell

Thesis/Purpose

To determine how modifications to adenosine affect binding to the adenosine deaminase active site

Long term goal: developing inhibitors to adenosine deaminase (drugs to treat diseases)

What is an enzyme?

catalyzes a chemical reaction Unique tertiary structure – active site

binds to specific substrates rate affected by temperature, pH, and

concentrations of both enzyme and substrate.

www.oak.cats.ohiou.edu

Enzyme and CatalysisP

oten

tial

En

ergy

Reaction Course

1. Transition State Stabilization

Inhibition of Enzymes Inhibitors: molecules that bind to enzyme and

slow down reaction Competitive Inhibitors – bind at same active site

as the substrate Noncompetitive Inhibitors – bind at a different

site• Changes enzyme shape, altering active site

www.eccentrix.com

Adenosine Deaminase (ADA)

Purine metabolism enzyme responsible for converting adenosine with water to inosine and ammonia.

http://sgc.utoronto.ca/SGC-WebPages/StructureDescription/2AMX.php

Adenosine Deaminase

ON

OHHO

N

N N

N

CH2OH

NH2 O

CH2OH

N

N N

NH

ON

OHHO

H2O

NH3

Adenosine Inosine

Did you know?

ADA imbalance within the body can result in a variety of health problems

High Levels of ADA activity are present in certain leukemias

Inhibition of ADA can stop growth of some cancerous cells

ADA and Coronary Artery Disease

Key role in immunity and inflammation Adenosine with active stress and hypoxia

balances oxygen supply Stimulates angiogenesis

www.medem.com

ReactionsMichaelis- menten equation:

Model:

Michaelis constant:

Vmax= k2[ Et]

Lineweaver-Burk:y = mx + b

www.nsr.bioeng.washington.edu

Adenosine Deaminase

ON

OHHO

N

N N

N

CH2OH

NH2 O

CH2OH

N

N N

NH

ON

OHHO

H2O

NH3

Adenosine Inosine

Beer’s Law

Absorbance (λ)= C · l · ε λ

Absorbance: amount of light absorbed at a definite wavelengthC: concentrationl: pathway (always one cm)ε λ: extinction coefficient: units = Abs

mol. · cm- absorbance of a given wavelength of light per

mole of a compound

www.biocompare.com

Experiment procedure1. Determine ε0 for adenosine

- Find optimum concentration (50um)

- Run baseline of H20, buffer (hepes), and 50 um inosine

- Scan 50 um adenosine from 220 to 300nm- Use peak in beer’s law 2. Find Vmax and Km

-Create 7 solutions using H20, HEPES, 10 uL of adenosine deaminase, and varying

adenosine concentrations

-run solutions at 264 nm and plot data

Monitoring rate of reactionKinet ic / Time: 15 um Adenosine

y = -0.0011x + 0.2268

R2 = 0.9773

0.222

0.223

0.224

0.225

0.226

0.227

0.228

0 1 2 3 4 5

Time (min)

Abs

Baseline Data

y = 3.09E+01x + 3.20E+06

R2 = 9.67E-01

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

0 100000 200000 300000 400000

1/ s0

1/

v0

Process Cont’d

determine if the derivatives are direct inhibitors: same procedure as for adenosine

Obtain optimal wavelengths and absorbencies and calculate ε for the 3 compounds

Compare reactivity with the enzyme for each (test for binding)

Create mixture of compound and adenosine, compare reactivity with original adenosine scan (Test for inhibition)

Adenosine and its Analogs

N

NN

N

NH2

O

OHOH

HH

HH

HO

N

NN

N

Cl

O

OHOH

HH

HH

HO

N

NN

N

NH2

O

OHOH

HH

HH

HO

N

NN

N

HN

O

OHOH

HH

HH

HOCl

Adenosine 6-Chloroadenosine

2-Chloroadenosine N6-Cyclohexyladenosine

6-Chloroadenosine

Kinetic/Time: 16um Chloro 10 minutes

y = -0.00023x + 0.15395

R2 = 0.97837

0.1515

0.152

0.1525

0.153

0.1535

0.154

0.1545

0 2 4 6 8 10 12

Time (min)

Abs

(mg/

mL)

AnalysisDouble Reciprocal Plot for 6-Chloroadenosine

y = 22.907x + 9E+06

R2 = 0.825

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

18000000

0 50000 100000 150000 200000 250000 300000 350000

1/[s]

1/[v

]

λ(nm) Abs ε (Abs/(M·cm))Vmax(M/s)

2.0x10-4 U/ml ADA

Km(µM)

Adenosine 264 0.315 6300 6.8x10-9 11.85

6-Chloroadenosine 269 0.105 2100 1.85x10-10 2.5

N6-CyclohexyladenosineTest for Inhibition with Cyclohexyladensine

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

0.004

0.0045

0.005

0 0.1 1 10 20 30

Concentration (um)

Reacti

on

Rate

(S

lop

e)

2-Chloroadenosine

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0.0014

5 10 20 30

Concentration 2-Chloroadenosine (uM)

Rate

Conclusion

Of the two positions we examined, the 2 position did not show evidence of binding, and

the 6 position showed possibility of binding, depending on the

substituent.

Future Goals…

Repeat/verify our results• More trials to determine uncertainty in our

values• different methods [liquid chromatography] to

compare results

Different modifications [n6 position] Varying environmental conditions

• pH• Temperature

The End

Thank you: Laura and John Overdeck Other Sponsors of NJGSS 2006 Director Miyamoto Surace Paul Quinn Myrna Papier Team Project Leader Dr. Cassano Team Project Assistant Jen Cowell