INHIBITION OF

SUCCINIC ACID

DEHYDRONASEBY:

RODAVIA, MARY MARJORIE

ROMERO, GENE PAUL

SAH. SITESH KUMAR

SALAGALA, JOHN PAUL

SALES , MICHELLE

SALTING, REYGIE

SALVADOR, ROMARIE JOY

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.

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.

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).

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

Pyruvic Acid Acetyl Co-A

Fatty AcidsFrom 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 DHSuccinyl Co-ASynthetase

SDH

Fumerase

MalateDH

NAD+

NADH+H+

NAD+

NAD+

NAD+

NADH+H+

NADH+H+

NADH+H+

ADPATP

FADH2

CO2

CO2

From Glycolysis

Kreb’s Cycle

FAD+

This occurs in the inner mitochondrial membrane by coupling the two reactions together.

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.

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.

The modern names are: malonate: propanedioate

succinate: butanedioate

fumarate: trans-butenedioateThe 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.

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

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.

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.

 

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.

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

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.

OBSERVATION AND RESULTS:

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:

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.

.

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.

Leigh syndrome