INHIBITION OF SUCCINIC ACID DEHYDRONASE

2. ENZYME
• Enzymes are protein catalysts that accelerate the
rates of biochemical reactions and regulate
metabolic pathways.
• They have extremely interesting properties that
make them little chemical-reaction machines.
• The purpose of an enzyme in a cell is to allow the
cell to carry out chemical reactions very quickly.
• These reactions allow the cell to build things or
take things apart as needed.
• This is how a cell grows and reproduces.
• At the most basic level, a cell is really a little bag
full of chemical reactions that are made possible
by enzymes.

3. Succinic Acid Dehydrogenase
 Also known as:
1. Succinate dehydrogenase
2. succinate-coenzyme Q reductase (SQR)
3. respiratory Complex II
 enzyme complex, bound to the inner mitochondrial membrane of mammalian mitochondria and
many bacterial cells.
 only enzyme that participates in both the citric acid cycle and the electron transport chain.

4. Succinate dehydrogenase
 Only makes the trans-fumarate.
 Donates electrons directly into complex II of the
respiratory chain (ubiquinone (Q)).
 If the respiratory chain is inhibited, FAD is unable
to accept electrons and TCA cycle stops.
 Inhibited by OAA, activated by coenzyme Q (part
of electron tranport chain).

5. DIFFERENT REACTION OCCUR IN THE CYTOPLASM
and we are going to study complex II.

6. Pyruvic Acid Acetyl Co-A
Fatty Acids
From Beta
Oxidation
Cis-Aconitic
Acid
Isocitric Acid
Alpha Keto-
Glutaric Acid
Succinyl Co-A
SUCCINIC ACID
Fumaric Acid
Malic Acid
Oxaloacetic Acid
PDH
PC
Citric Acid
CS
IDH
AKG DH
Succinyl Co-A
Synthetase
SDH
Fumerase
Malate
DH
NAD+
NADH+H+
NAD+
NAD+
NAD+
NADH+H+
NADH+H+
NADH+H+
ADP
AT
P
FADH2
CO2
CO2
From Glycolysis
Kreb’s Cycle
FAD+
This occurs in the inner
mitochondrial membrane
by coupling the two
reactions together.

7. Enzyme inhibition
An enzyme inhibitor is a molecule
that binds to enzymes and
decreases their activity. Since
blocking an enzyme's activity can
kill a pathogen or correct a
metabolic imbalance, many drugs
are enzyme inhibitors.

8. Competitive inhibitors
 This is the most straightforward and obvious form of enzyme inhibition - and the
name tells you exactly what happens.
 The inhibitor has a similar shape to the usual substrate for the enzyme, and competes
with it for the active site. However, once it is attached to the active site, nothing
happens to it. It doesn't react - essentially, it just gets in the way.
 The complex doesn't react any further to form products - but its formation is
still reversible. It breaks up again to form the enzyme and the inhibitor molecule.
 That means that if you increase the concentration of the substrate, the substrate can
out-compete the inhibitor, and so the normal reaction can take place at a reasonable
rate.
 A simple example of this involves malonate ions inhibiting the enzyme succinate
dehydrogenase. This enzyme catalyzes the conversion of succinate ions to fumarate
ions.

9. The modern names are:
 malonate: propanedioate
 succinate: butanedioate
 fumarate: trans-butenedioate
The conversion that succinic dehydrogenase carries out is:
The reaction is inhibited by malonate ions which have a very similar shape to succinate ions.
Succinic acid
-Also known as butanedioic acid; historically known as spirit of amber
-a white, odorless solid. Succinate plays a role in the citric acid cycle, an energy-yielding
process.

10. Malonate :
o a competitive inhibitor of the enzyme succinate dehydrogenase:
o is a structural analogue of succinate
o binds to the active site of the enzyme without reacting, and so competes with
succinate, the usual substrate of the enzyme.
o The observation that malonate is a competitive inhibitor of succinate
dehydrogenase was used to deduce the structure of the active site in that enzyme.
COO-

CH2

COO-
Malonate
COO-

CH2

CH2

COO-
Succinate

11. The similar shape lets the malonate ions bind to the active site,
but the lack of the CH2-CH2 bond in the centre of the ion stops any
further reaction taking place.
The malonate ions therefore block the active site - but remember that this is
reversible. The malonate ions will break away and free up the enzyme again.
The malonate ions are in competition for the site - they aren't destroying it.
If the succinate ions have a greater concentration than the malonate ions,
by chance they will get access to the site more often than the malonate ions.
That means that you can overcome the effect of a competitive inhibitor by
increasing the concentration of the substrate.

12. Non-competitive inhibitors
 A non-competitive inhibitor doesn't attach itself to the active site, but attaches somewhere else on
the enzyme. By attaching somewhere else it affects the structure of the enzyme and so the way the
enzyme works. Because there isn't any competition involved between the inhibitor and the substrate,
increasing the substrate concentration won't help.

13. EFFECT OF INHIBITOR
 Few enzymes have absolute specificity for a single substance. Frequently,
they are compounds other than the usual substrate that when combine with
the enzymes, the enzymes does not react further to form the product. The
reaction is slowed or stopped according to the enzyme molecule that is
blocked.
 Inhibition due to structural similarity is shown in the case of succinate
dehydrogenase, which catalyzes the oxidation of succinic acid to fumaric
acid. In the presence of malonic acid, the action of succinate dehydrogenase
is almost completely inhibited.

14. PREPARATION OF SUCCINIC
ACID DEHYDROGENASE
• Dissect a rat and perfuse the intact liver
with saline.
• Remove the perfuse liver and weigh.
• Cut the whole liver into small pieces with
the aid of pair of scissors and
homogenize with enough 0.1 M
phosphate buffer, pH 7.4.
• Centrifuge and collect the sediment
(particulate fraction).
• Suspend the particulate fraction in
sufficient water to make 1:2 ratio with
the original weight of the liver.
EXPERIMENT 1: FACTOR THAT INFLUENCE ENZYMATIC ACTIVITY

15. REAGENT ADDED TUBE NO.
1 2 3
0.02% Methylene blue 0.5 ml 0.5 ml 0.5 ml
1 M Phosphate Buffer 0.5 ml 0.5 ml 0.5 ml
0.1 M Sodium Succinate 0.2 ml 0.2 ml ----
0.1 M Sodium Malonate ---- 0.3 ml 0.3 ml
Distilled Water 0.3 ml ---- 0.2 ml
Particulate Fraction 0.5 ml 0.5 ml 0.5 ml
PROCEDURE: Set up the following in # test tubes:
After the particulate fraction has been added, immediately layer each tube
with 1 ml of mineral oil and allow the tubes to stand undisturbed for one
hour. Record your observation.

16. OBSERVATION AND RESULTS:

17. OBSERVATION AND RESULTS:
1.Write down the chemical structure of malonate and succinic
acids. Give a rational explanation of the inhibitory effect of
malonic acid on the enzymes.
2.Is malonic acid dehydrogenated?
QUESTIONS:

18. WRITE DOWN THE CHEMICAL STRUCTURE OF
MALONATE AND SUCCINIC ACIDS. GIVE A
RATIONAL EXPLANATION OF THE INHIBITORY
EFFECT OF MALONIC ACID ON THE ENZYMES.
 The inhibition of succinate oxidation by malonate is a well known
phenomenon. Since the oxidation of succinate to fumarate is integral
part of the Krebs cycle of oxidations, it has been generally assumed
that the inhibitory effect of malonate upon the oxidation of any
member of the cycle is the result of the inhibition of the succinate to
fumarate step. They have similar shape to the substrate, so compete
the bond to the enymes active site.

19. IS MALONIC ACID DEHYDROGENATED?
• Succinic dehydrogenase having combined with malonic acid rather than
succinic acid can neither dehydrogenate it or readily lose it again. The
enzyme site is thus blocked. The process may be reversed by adding an excess
of succinic acid which competes with malonic acid for the action site.

20. Leigh syndrome