Enzyme Inhibition

Results
Composition of incubation mixture of Ach:
Reagents

Conc. required

Volume added

Buffer

0.05M

2.5ml

AChI

0.5mM

0.5ml

DTNB reaent

0.2mM

0.5ml

Acetylcholinesterase

0.05 units

0.5ml

H2O

N/A

1.0ml

Composition of incubation mixture of Ach + inhibitor (n-pentyl TMA):

Reagents

Conc. required

Volume added

Buffer

0.05M

2.5ml

AChI

0.5mM

0.5ml

DTNB reaent

0.2mM

0.5ml

Acetylcholinesterase

0.05 units

0.5ml

n-pentyl TMA

0.05mM

0.5ml

H2O

N/A

0.5ml

Data without the presence of n-pentyl TMA:

Ach Final Conc.
(mM)

Init. Velocity
(absorbance
change / min)

1 / V (min)

1 / S (min-1)

0.05

0.0186

53.76

20

0.1

0.0463

21.60

10

0.2

0.0785

12.74

0.3

0.1147

8.72

3.33

0.5

0.1237

8.08

Data with the presence of n-pentyl TMA:

Ach Final Conc.
(mM)

Init. Velocity
(absorbance
change / min)

1 / V (min)

1 / S (min-1)

0.05

0.0223

44.84

20

0.1

0.0345

28.99

10

0.2

0.0584

17.12

0.3

0.0841

11.87

3.33

0.5

0.0979

10.21

Ki for n-pentyl TMA with Ach of my group:

x-intercept of the Lineweaver-Burk plot without n-pentyl TMA: -3.4
x-intercept of the Lineweaver-Burk plot with n-pentyl TMA:
-2.4
Km for ACh = 1 / 3.4 = 2.9 x 10-4M
1+ [I] / Ki = -3.4 / -2.4 = 1.417
[I] / Ki = 0.417
As [I] = 0.05mM
Ki = [I] / 1.125 = 0.05 x 10-3 / 0.417 = 1.2 x 10-3M
Ki for n-pentyl TMA with ACh:
1.5 x 10-4M
6.5 x 10-4M
2.3 x 10-4M
1.8 x 10-4M
1.3 x 10-4M
7.7 x 10-4M

Average value: 3.5 x 10-4M

Ki for phenyl TMA with ACh:

1.43 x 10-5M
3.00 x 10-5M
5.90 x 10-5M
2.70 x 10-5M

Average value: 3.25 x 10-5M

Ki for edrophonium with ACh:

3.3 x 10-8M
9.3 x 10-8M
3.0 x 10-7M

Average value: 1.4 x 10-7M

Write-up
Brief notes of of the three inhibitors
Edrophonium is a short-acting anticholinesterase. Anticholinesterases are compounds
that block the hydrolysis of neurotransmitter acetylcholine in the synapse by
inhibiting acetylcholinesterase and therefore prolong its action. They enhance
neuromuscular transmission but may also have muscarinic effects. They have various
pharmacological actions and therapeutic uses. Edrophonium can be classified as a
competitive reversible inhibitor of acetylcholinesterase. It is a quaternary ammonium
compound that binds to the anionic site of acetylcholinesterase only. The ionic bound
formed is readily reversible and the action of the drug is short in vivo. It is used
mainly for the diagnosis of myasthenia gravis.
n-pentylTMA and phenylTMA are also competitive inhibitors of acetylcholinesterase.
Mechanism of acetylcholinesterase-ACh interaction
Its active site has two parts:
1. the esteric site which lines up with the carbonyl group of ACh
2. the anionic site that has a negative charge lines up to bond the positively charged
nitrogen
The active part of the enzyme contains a reactive serine as part of a catalytic triad
together with a histidine and an aspartic acid. These three amino acids are far apart in
the primary structure but they are much closer by the folding of polypeptide chain.
Such arrangement allows the transfer of a proton from serine to histidine. This
activates the serine for the attack by the carbonyl carbon atom of ACh. The tetrahedral
intermediate accepts protons from histidine and releases the amine. The acetylated
enzyme is then hydrolyzed, releasing the carboxylic acid and regenerating the
enzyme.
Comparison of Km of ACh and Ki of edrophonium
It is a conformationally restricted analogue of choline, which cannot change shape by
rotation of bonds that easily thus has a higher affinity for the enzyme than ACh does.
It can fit well in the active site. This account for the much lower Ki (1.4 x 10 -7M) of
edrophonium than the Km of ACh (2.9 x 10 -4M), as Km and Ki is inversely
proportional to the affinity of enzyme-substrate and enzyme-inhibitor interaction,
respectively.
Structure-activity relatioinships
The key features of ACh molecule that are important for its binding to

Acetylcholinesterase are the quaternary ammonium group which is positively charged

and the positively charged ester group which is susceptible to rapid hydrolysis by
cholinesterase.
The three inhibitors all got quaternary ammonium group but not ester group with
respect to the structure of ACh. They will bind to the anionic site hence block the
binding of ACh to the active site. Thus break down of ACh is hindered. With the
exception of edrophonium that is a conformationally restricted analogue of choline, npentylTMA and phenylTMA bind noncolvalently to the enzyme and the strength of
binding is of a similar order to the substrate, which means that Km of n-pentylTMA
and phenylTMA are of similar size to Km of ACh. PhenylTMA has a higher lower Ki
value as it can fit better to the active site with respect to n-pentylTMA.
Dose of edrophonium
Concentration required = Ki * 10 = 1.4 x 10-7M * 10 = 1.4 x 10-6M
Volume required = 1.4 x 10-6M * 41 = 5.74-5mole
Mass required = RMM * 5.74-5mole = 9.53mg
For the detection of myasthenia gravis in adult, it requires 2mg followed after
30seconds by 8mg by intravenous injection and it requires 10mg by intramuscular
injection. Mass calculated above is similar to the dose in BNF for the detection of
myasthenia gravis.
For the detection of myasthenia gravis in child, 20mcg/kg followed after 30 seconds
by 80mcg/kg is required. Lets say the resultant dose is same as the calculated
one(9.53mg). Mass of the child is:
9.53 * 1000 / (20 + 80) = 95.3kg which is not a realistic value for the weight of a
child. Thus the calculated mass is too high when comparing with the dose.