A competitive inhibitor of succinic dehydrogenase is
Answers
Answer:
Enzymes can be activated or inhibited in different ways. Aspecific inhibition is possible by a drastic change in
pH, temperature or ionic concentration. Much more information can be gained by using specific enzyme inhibitors.
Such enzyme kinetic analysis may shed light on the mechanism of enzyme activity.
Competitive inhibitor binds reversibly to the free enzyme, the enzyme-inhibitor complex has no catalytic
activity and thus less active enzyme is available for interaction with the substrate. This effect is reflected in the increase
of the apparent Michaelis constant (Km), because more substrate is required to achieve the half-maximal reaction rate.
At extremely high (S>>Km) substrate concentration the inhibition becomes negligible, the Vmax is not changed. Either the
substrate or the inhibitor is bound to the enzyme, not both of them.
E + S ↔ ES→ E + P
+
I
↕
EI
On the basis of the aforementioned definition we can see that competitive inhibition is only a characteristic of
kinetic behaviour of the enzyme, but does not say anything about the structural background of the interaction of
inhibitor and enzyme, about the binding site of the inhibitor, which can be either the active site or an allosteric site.
Historically the first described competitive inhibitors were structural analogues of the substrates that bind to the active
site instead of the substrate, Malonate used in the present experiment is also a substrate analogue.
Succinate dehydrogenase (SDH) is the only inner mitochondrial membrane-bound enzyme of the citric acid
cycle, the rest of the enzymes are found in the matrix in a soluble form. Furthermore, SDH comprises the second
complex (complex II) of the respiratory chain. Only this complex has not proton-pumping activity. The enzyme contains
iron-sulfur centers as electron acceptors, a FAD prosthetic group which serves as hydrogen acceptor in the following
reaction:
COOCH2
CH2
COOE-FAD
COOCH
C
COOH
FADH2
+ +
Succinate Fumarate
In this experiment we apply different concentrations of succinate as a substrate, in the presence and absence of the
inhibitor malonate (a dicarboxylic acid with three-carbons). While in mitochondria the electrons from complex II are
transferred to ubiquinone (coenzyme Q), in our experiment we use an artificial acceptor, iodonitrotetrazolium chloride
instead, to which SDH will transfer the two electrons. The oxidized form of iodonitrotetrazolium (which is a redox dye)
is colourless; the end product is reduced iodonitrotetrazolium formazan, which is red coloured. The absorbance (A) of
the solution is directly proportional to the concentration of the coloured compound formed (cformazan), which reflects
the enzyme activity, so iodonitrotetrazolium formazan formation can be followed spectrophotometrically. A = ε c l,
where ε = 0.0184 cm-1
M
-1 a proportionality constant that is typical for formazan, it equals the absorbance of 1 μM
formazan when the length of the lightpath in the solution is 1 cm (its old name is micromolar extinction coefficient),
and l = 1 cm, the lightpath in our photometric cell. The reaction is shown below:
2
succinate FAD iodonitrotetrazolium formazan (red, reduced)
fumarate FADH2 iodonitrotetrazolium chloride (oxidized, colourless)
Solutions:
1.) mitochondrial membrane:,10 mg/ml protein in KCl-TRIS buffer pH, 7.4 [TRIS: tris(hydroxymethyl)aminomethane]
2.) 0.5 M phosphate (KH2PO4 + K2HPO4) buffer, pH 7.6
3.) 125; 500 mM succinate
4.) 250 mM malonate
5.) 0.5 % iodonitrotetrazolium chloride (INT)
6.) formate-Triton-formaldehyde solution (FTF)
Set up the experiment according to the table below.
In 11 numbered test tubes pipette the following components in the written order.
1 2 3 4 5 6 7 8 9 10 C
phosphate buffer μl 360 340 360 340 280 340 320 340 320 260 280
125 mM succinate l 20 40 - - - 20 40 - - - -
500 mM succinate l - - 20 40 100 - - 20 40 100 -
250 mM malonate l - - - - - 20 20 20 20 20 -
INT l 100 100 100 100 100 100 100 100 100 100 100
FTF (ml) - - - - - - - - - - 3
SDH (mitoch. mb) l 20 20 20 20 20 20 20 20 20 20 20
incubation for 10 min at 37 :C in waterbath
FTF ml 3 3 3 3 3 3 3 3 3 3 ---
3 ml FTF is added from the dispenser bottle. In FTF, formate and formalin (aqueous solution of formaldehyde)
denature the proteins, therefore they block the enzyme activity. Triton is a detergent, it solubilizes the membrane and
the hydrophobic formazan, and therefore subsequent spectrophotometric measurement is possible without filtration
or centrifugation.
Set the spectrophotometer to measure absorbance at 490 nm. Use the solution in the C-marked tube as 'blank'
to calibrate the spectrophotometer (to set the reference value of zero absorbance).