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Enzyme Inhibition: Aims

Review of Practicals

To demonstrate the digestions of caesin


by protease enzymes

BIOC2101

To provide an understanding of how an


enzyme may be inhibited initially as a
result of synthesis as a zymogen

Enzyme Inhibition: Aims


To provide an understanding of how the
zymogen precursors of protease
enzymes are activated in the small
intestine

Background
Parts A, B and C all use an assay for
protease activity
The substrate is casein
The enzyme is a pancreatic protease

To illustrate the specific inhibition of


proteases and the role of pancreatic
trypsin inhibitors

Background
The protease hydrolyses internal bonds
within the casein to produce smaller
peptides
This reaction can be stopped by adding
TCA, which denatures and precipitates
the proteins (enzyme and large casein
molecules)
Centrifugation ensure only the small
peptides remain in the supernatant

Background
Bradford's reagent is used to measure
the amount of peptides present in the
supernatant
The reagent produces an intense blue
colour with all peptides and proteins
(measured at 620 nm)

Part A

Part A

Substrate and
buffer
RO water

Trypsin inhibitor

Trypsin

Trypsin

0 min 10 min 20 min 30 min

Trypsin inhibitor acts to inhibit the


protease activity of trypsin

0 min 10 min 20 min 30 min

Reactions stopped
Bradford assay performed

Part B
Substrate and
buffer
RO water

Trypsin inhibitor

chymotrypsin

chymotrypsin

0 min 10 min 20 min 30 min

Part B
Trypsin inhibitor does not inhibit the
protease activity of chymotrypsin

0 min 10 min 20 min 30 min

Reactions stopped
Bradford assay performed

Part C
Chymotrypsinogen is a zymogen
Chymotrypsinogen is activated by
trypsin

Part C
chymotrypsinogen
trypsin
trypsin
0 min 5 min 10 min
inhibitor

15 min 20 min 35 min

Casein added
Reactions stopped
Bradford assay performed

Glycolysis(Prac,cal(

Part C
The longer the incubation, the more
chymotrypsinogen is activated
The trypsin inhibitor stopped the
zymogen from being activated and
prevents trypsin from acting on the
casein
The more chymotrypsin is present, the
more casein will be digested

GLUCOSE
ATP

Provided with:-"
- Dialysed rat skeletal muscle extract, containing all the
enzymes for glycolysis and other soluble cytoplasmic
enzymes, but no mitochondrial enzymes and no
metabolites/cofactors."
"
- Assay system for lactate"
"
Expt A investigated what cofactors are necessary for
glycolysis (fructose-1,6-bisP --> lactate)"
"
Expt B investigated the rate limiting step in glycolysis
(glycolytic intermediate --> lactate)"

GLUCOSE
ATP

Experiment A

ADP
G-6-P
F-6-P
ATP

F-6-P
ATP

ADP
F-1,6-bisP

DHAP

ADP
F-1,6-bisP

GAP

Expect to find
that ADP, Pi
and NAD+ are
all required for
rapid glycolysis"

NAD+ + Pi

NADH + H+
2 x 1,3-bisPG
ADP
ATP
2 x 3-PG

DHAP

NAD+ + Pi

ATP
2 x 3-PG
2 x 2-PG

H2O
2 x PEP
ADP

NADH

ATP
2 x PYR

H2O
2 x PEP
ADP

"

NAD+

lactate

GLUCOSE
ATP

F-6-P
ATP
ADP
F-1,6-bisP

NAD+ + Pi

NADH + H+
2 x 1,3-bisPG
ADP
ATP
2 x 3-PG
2 x 2-PG
H2O
2 x PEP
ADP

Why was there some


glycolysis in the
absence of Pi?!
!
Phosphatases, e.g.!
ADP --> AMP + Pi!
(and phosphate
release from AMP
and NAD+)"

GLUCOSE
ATP

lactate

ADP
G-6-P

"

"

lactate

"

Experiment A

F-6-P
ATP

Why might there be


some glycolysis in the
absence of NAD+?!
GAP
NAD+ + Pi !
NADH + H+ NAD+ binds very
2 x 1,3-bisPG
ADP
tightly to some
ATP
enzymes and is
2 x 3-PG
difficult to remove
2 x 2-PG
completely by dialysis!
H2O

ADP
F-1,6-bisP

DHAP

2 x PEP
ADP

"

NADH NAD+

ATP
2 x PYR

Typical Results"
"
Addition !
!A"
Water
"
"0.02"
NAD+/ADP/Pi "0.45"
NAD+/ADP"
"0.10"
NAD+/Pi "
"0.02"
ADP/Pi
"
"0.04"
""

NADH NAD+

ATP
2 x PYR

"

Experiment A

ADP
G-6-P

GAP

GAP

NADH + H+
2 x 1,3-bisPG
ADP

2 x 2-PG

DHAP

Experiment A

ADP
G-6-P

ATP
2 x PYR

"

NADH NAD+

lactate

"

GLUCOSE
ATP

GLUCOSE
ATP

Experiment B

ADP
G-6-P
F-6-P
ATP

F-6-P
ATP

ADP
F-1,6-bisP

ADP
F-1,6-bisP

DHAP

GAP

Start with:-"
Glucose"
F-6-P"
F-1,6-bisP"
GAP"
1,3-bisPG"
+ all necessary
cofactors"

NAD + Pi

NADH + H+
2 x 1,3-bisPG
ADP
ATP
2 x 3-PG
2 x 2-PG
H2O
2 x PEP
ADP

NADH

ATP
2 x PYR

DHAP

GAP

2 x 2-PG
H2O
2 x PEP
ADP

"

NAD+

ADP
F-1,6-bisP

Why was lactate


formed starting from
glucose and F-6-P
GAP
NAD+ + Pi when no ATP was
NADH + H+ added?!
2 x 1,3-bisPG
ADP
!
ATP
Adenylate kinase!!!
2 x 3-PG
2ADP <--> AMP + ATP!
2 x 2-PG
H2O

NADH

"

pyruvate"

2 x 2-PG

"

NADH NAD+

"

How(can(lactate(dehydrogenase(catalyse(reac,ons(in(the(
opposite(direc,ons(during(rst(glycolysis(and(then(the(lactate(
assays?(
Glycolysis"
NADH + H+"

DHAP

Why was NO lactate


formed starting from
1,3bisPG?!
GAP
NAD+ + Pi !
X!NADH + H+ NO NADH was
2 x 1,3-bisPG
ADP
produced to reduce
ATP
pyruvate to lactate!!!
2 x 3-PG
H2O
2 x PEP
ADP

NAD+

lactate

Experiment B

ADP
G-6-P

ADP
F-1,6-bisP

ATP
2 x PYR

"

lactate"

GLUCOSE
ATP

F-6-P
ATP

2 x PEP
ADP

Typical Results"
"
Addition !A"
Water
"0.02"
Glucose "0.08"
F-6-P"
"0.15"
F-1,6-bisP "0.45"
GAP "
"0.50"
1,3-bisPG "0.02"

NADH NAD+

ATP
2 x PYR

F-6-P
ATP

DHAP

NAD+ + Pi

ATP
2 x 3-PG

Experiment B

ADP
G-6-P

rate
limiting
step"

NADH + H+
2 x 1,3-bisPG
ADP

lactate"

GLUCOSE
ATP

Experiment B

ADP
G-6-P

Relatively low pH
of 7.4 (high [H+])
lactate" favours lactate
formation"

NAD+"

Lactate assay"

Relatively high pH
of 9.4 (low [H+])
NAD+" NADH + H+"
lactate"
pyruvate" favours pyruvate
formation"
reacts non-enzymically
with hydrazine"

ATP
2 x PYR

lactate

"

Separa,on(Techniques(
3 types of chromatography:-"
"
Partition (thin layer)"
"
Permeation (gel filtration)"
"
Ion exchange"
"
All involve the differential distribution of
molecules between a stationary phase
and a mobile phase"

TLC(separa,on(of(amino(acids(
The Mobile phase is the solvent (n-butanol/acetic acid/
water).!
!
The Stationary phase is water associated with
cellulose fibres."
"
More polar molecules partition into the stationary
aqueous phase and do not migrate far from the origin.
(e.g. lysine and glutamic acid)"
"
Less polar molecules are more soluble in the organic
mobile phase and migrate further. (e.g. leucine)"

What(is(the(func,on(of(the(cellulose(in(
the(TLC?(

Provides a solid insoluble support for


the stationary phase , which is water
associated with the cellulose."

Results(of(the(Gel(ltra,on(Expt.(
Yellow ferricyanide
forms a diffuse slow
moving band!

Red/brown
hemoglobin forms a
much tighter fast
moving band!

Why(does(the(Rf(change(in(dierent(
solvents?(
Different solvents may be more or less polar, affecting
partitioning between the stationary and mobile phases."
"
Different solvents may change the ionisation of amino
acids and therefore their distribution between polar
stationary and less polar mobile phases."
"
e.g. glutamate is less polar in an acidic solvent than in
a basic solvent."
-OOC-CH

+
2-CH2-CH(NH3 )COO "
HOOC-CH2-CH2-CH(NH3+)COO-"
HOOC-CH2-CH2-CH(NH3+)COOH"

Gel(ltra,on(
The Mobile phase is the buffer which passes through
the chromatography column.!
!
The Stationary phase is water (buffer) immobilised in
pores in the gel filtration particles, that is accessible to
relatively small molecules, but not to large molecules."
"
Larger molecules will pass through the column faster
than smaller molecules (the opposite of conventional
filtration)."

Ion(exchange(chromatography(
The Mobile phase is the buffer which passes through
the chromatography column.!
!
The Stationary phase is ionised groups immobilised
on the ion exchange particles."
"
Molecules with net charges opposite to those on the ion
exchange column will form ionic bonds and bind tightly
to the stationary phase. Molecules with no net charge
(or with a net charge the same as that on the ion
exchange column) will pass through with the mobile
phase."

Ion(exchange(chromatography(

Ion(exchange(chromatography(

The Stationary phase has negatively charged


groups immobilised on the ion exchange particles"
(-SO3-)."
"
Glycine has no net charge at pH 5.2"
"
"+H3N-CH2-COO-"
BINDS!
"
Arginine has a net charge of ~+1 at pH 5.2"
"
"+(NH2)2C-NH-CH2-CH2-CH2-CH(NH3+)-COO-"
"

At pH 2.0!
"
Glycine has a net charge of ~+0.5 "
"
"+H3N-CH2-COO- and +H3N-CH2-COOH"
"
Arginine has a net charge of ~+1.5"
"
"+(NH2)2C-NH-CH2-CH2-CH2-CH(NH3+)-COO-"

Ion(exchange(chromatography(of(proteins(
Ion exchange chromatography is an important
technique for separating and purifying proteins.!
"
The pI (isoelectric point or isoelectric pH) is the pH at
which a protein has an overall charge of zero."
"
This means that at pH values <pI, a protein will have an
overall +ve charge, and bind to an ion exchange column
which has -ve charged groups."
"
At pH values >pI, the protein will have an overall -ve
charge and bind to an ion exchange column which has
+ve charged groups."

Glucose(Tolerance(Test(Prac,cal(

"

and "+(NH2)2C-NH-CH2-CH2-CH2-CH(NH3+)-COOH"
"
BOTH will bind, but the arginine will elute much more
slowly."

Glucose(Tolerance(Test(Prac,cal(
16
14
12

Patient 1

10

Patient 2

Patient 3

Patient 4

Patient 5

normal!
onset of
diabetes!
untreated
diabetic!

0
0

30

60

90

120

150

180

Time (min)

Someone with untreated diabetes is expected to have a high fasted blood


[glucose] (time zero), a very high blood [glucose] after taking the glucose
load; and a blood [glucose] that has not returned to normal values after 3 h."

What(other(tests(might(assist(the(
diagnosis?(

16
14
12

Patient 1

10

Patient 2

Patient 3

Patient 4

Patient 5

normal!

No fast!

2
0
0

30

60

90

120

Time (min)

150

180

Urinary [glucose]"
"
Plasma [insulin]"
"
Glycosylated hemoglobin"

Fasting is required to
ensure a normal
initial fasted blood
glucose"

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