Mitochondrial Complex I Inhibitor Rotenone Induces Apoptosis through Enhancing Mitochondrial Reactive Oxygen Species Production

References

Li, Nianyu , Kathy  Ragheb, and Gretchen  Lawler. “Mitochondrial Complex I Inhibitor Rotenone Induces Apoptosis through Enhancing Mitochondrial Reactive Oxygen Species Production.” The Journal Of Biological Chemistry 278, no. 10 (2003): 8516-8525. Accessed April 13, 2013. http://www.jbc.org/content/278/10/8516.full#sec-22

 PUBLISHED PAPER 2

Rotenone inhibits complex 1 in the electron transport chain (ETC) and has leads to the death of a number of cells. The reactive oxygen species (ROS) aids in apoptosis and the inhibiting of complex 1 by rotenone promotes the creation of ROS. Rotenone along with the increased ROS generates a similar substrate to complex 1 substrate. DNA fragmentation was able to determine that apoptosis, the release of cytochrome c and caspase is caused by rotenone. Apoptosis caused by rotenone can be stopped using antioxidants. In apoptosis, magnesium superoxide dismutase is resilient to rotenone-induced mitochondrial ROS.

 

Complex 1- 4 and ATP synthase in the ETC produces ATP as well as assist in apoptosis of the cell due to the ROS. Mitochondria discharge a number of proapoptotic regulators such as cytochrome c to the cytosol when the apoptosis process is stimulate. The regulators triggers the apoptotic process, as well as it is controlled by proteins and results in the mitochondria being the central path of apoptosis signal.

 

The significance of rotenone-induced apoptosis was determined with a tumor necrosis factor (TNF)-α which can disrupt complex 1 in ETC while other intelligences state that rotenone can inhibit cells outside the mitochondria.

 

ROS is suspected to be activated by several other molecules within the body including UV radiation. ETC produces the main supply of ROS under biological conditions and hence it is been thought that ROS formed from the mitochondria contributes to the apoptosis process. The ROS mitochondria manipulate the mitochondria functioning exclusive of the cytosol, two complexes in the ETC, complex 1 and 3 are also known to be contributors of ROS

 

From chemical determination superoxide and hydrogen peroxide was determined to be primary and secondary products and it has been noted that rotenone as well as complex b-c1 inhibitor antimycin can cause the formation of both oxides. The ROS made in the mitochondria can be altered when inhibition takes place.

Biochem crossword

K

 

ACROSS

1. A pocket or a cleft

2. Maximal velocity

3. Removal of water

4. S shaped curve

5. Covalent bond formed between the alpha amino group and the alpha carboxyl group

6. Smallest unit that is capable of performing life functions

7. Change in the optical rotation that occurs by epimerization

8. Two sugars that differ only in the configuration around one carbon atom

9. Common description of denaturation

10. Five membered ring

11. Each of two or more enzymes with identical function but different structure

12. Apoenzyme + cofactor

13. Has an aldehyde group

14. Reaction between a aldehyde and an alcohol

15. pH at which Amino acid would not move in an electric field

16. A substance that diminishes the velocity of an enzyme catalyzed reaction

DOWN

1. Inactive protein part

3. Have the same number of atoms arranged differently in space

6. Hydrates of carbon whose main role is to provide energy

12. When the substrate itself serves as an effector

14. Reaction between a ketone and an alcohol

17. Highest arrangement of atoms that is in the intermediate structure between reactants and product

18. A non-protein compound necessary for the functioning of an enzyme.

19. Enzymes that are composed of more than one sub-units

20. Has a keto group

21. One of two stereoisomers of a cyclic saccharide that differs only in its configuration at the hemiacetal or hemiketal carbon

22. Six membered ring

23. The amount of energy that is needed to convert all the molecules in one mole of a reacting substance from a ground state to the transition state.

24. A monosaccharide sugar that contains four carbons

25. Michaelis constant

26. Biological catalyst that speed up a chemical reaction by providing an alternate pathway with a lower activation energy

27. Vo = Vmax / Km + [S]

28. A molecule or ion having separate positively and negatively charged groups

29. A molecule that cannot be synthesized by the body and is obtained from the diet

30. Dynamic Equilibrum

31. A covalent bond that joins a sugar molecule to another sugar

32. 1 / micrometer per minute

 

Anticholinesterase Inhibition – Published Paper Review

Weinbroum, Avi A. “Pathophysiological and clinical aspects of combat anticholinesterase poisoning.” British Medical Bulletin 72, no. 1 (2004): 119-133. Accessed March 24, 2013.  http://bmb.oxfordjournals.org/content/72/

 

Anticholinesterase (AChE) is an enzyme and like all enzymes, their function is affected by several factors.

RECALL: Enzymes are affected by pH, temperature, substrate and enzyme concentration and inhibitors. These agents tend to disrupt the area of the enzyme called the active site by changing its conformation and hence hindering enzymatic activity.

INTRODUCTION

Organophosphate – like compounds are inhibitors of the enzyme acetylcholine esterase (AChE) and are referred to as nerve agents (NAs). It is related to those used in pesticides. The extremely lethal irreversibly AChE inhibitors results in an inoperative AChE causing a buildup of acetylcholine (ACh) affecting the entire cholinergic system.

DID YOU KNOW

In the 1854, Wurtz synthesized the first organophosphate compound, tetraethyl pyrophosphate.

Sad to say…. organophosphates were used as weapons of mass destruction

1980 Iraq – Iran war

1994-95 – Terrorist attacks in Japan

CLINICAL ASPECTS

The extent of absorption and contact with NA are vital issues in verifying biochemical intoxication route for the NA. NA can either be absorbed by the skin and mucous membrane or inhaled. The inhalation of the vapor results in instantaneous absorption due to the alveoli in the lungs causing respiratory ailments ranging from short breath to cardiovascular and respiratory failure and eventually death. Exposure to the skin has a nicotine-like effect causing muscle paralysis and continued developing respiratory indications and ultimately death.

BIOCHEMISTRY OF NAs

They are chemically obtained from phosphoric acids. They tend to differ slightly by substituting the -OH radical and the acid-base form but are still efficient of being both reversible and irreversible inhibitors to AChE. NAs chemical properties are such that it is both colorless and odorless in its volatile liquid state and denser that air in its gaseous state.

AChE enzyme is categorized as class three, Hydrolase and sub-class as esterase and therefore from the class it is determined that the enzyme hydrolyses esters, especially choline esters, ACh, a neurotransmitter for the cholinergic part of the nervous system.

AChE hydrolyses ACh swiftly and is located in the receptors sites of the nerves by the cholinergic nervous system. Reversible anticholinesterases are not lethal as compared to the irreversible anticholinesterases. Carbamates bind to anionic and the ester of the enzyme resulting in the separation of part of the carbamate and the formation at the location of the ester, an enzyme-cabamylated complex. Hence hydrolysis of ACh is no longer a rapid process. The most attractive binding sites for organophosphate compounds are the ester sites however the stability of the bonds depend on the correct orientation of the enzyme and inhibitor as well as other compounds are prevented from binding with the active site. They interfere with the cleft leaving the enzyme dysfunctional.

With irreversibly inhibition the NAs covalently binds (strongest bond formation) to the active site so therefore it is known as a completive inhibitor. This result in a buildup of the ACh in the neuro-effector junction hindering the synapse process in peripheral and the central cholinergic system causing toxic contamination to the nicotinic (CNS) and muscarinic cleft (muscle system). The covalent bond partakes in an instantaneous chemical reaction which stabilizes the molecule due to the surge in the thermodynamic stabilization resulting in the production of more hydrogen bridges between the phosphate and organic groups.

The skeletel muscles, the pre-ganglionic autonomic nerves and the post-ganglionic parasympathetic nerves are innervated by AChE. The cholinergic systems are bases on the muscarinic and nicotinic systems since they have receptors that display specificity to muscarinic alkaloids and nicotine alkaloids. The post-ganglionic parasympathetic fibers are innervated by the muscarinic sites which regulate the activity of the glands, smooth muscle of the respiratory, cardiovascular and gastrointestinal systems. Autonomic ganglia, part of the nicotinic sites are relied upon for the contractions of the skeletal muscle. When both muscarinic and nicotinic cholinergic neurotransmitter buildup it causes hyper-stimulation of the synaptic process and hence causes paralysis of the skeletal muscles. Comparably the buildup of AChE in the CNS nerve receptors leads to hyper-stimulation and paralysis cardiac brady-asystole, hyper-secretion from secretory glands, respirational collapse, seizures, coma and eventually it is fatal. A small window of opportunity allows antidote drugs, atropine and oximes to work against the inhibitor stimulated nicotinic and muscarinic cholinergic system correspondingly depending on the stage of poisoning.

CONCLUSION

NAs are highly poisonous complexes that result in fatality within mere seconds. The principal biochemical cause NAs is the capability to irreversible inhibit AChE enzyme resulting in the buildup of ACh in the synaptic cleavage. Antidotes such as atropine and oximes present a crucial windowed-opportunity to act against the inhibitor

 

 

Enzyme Browning Reaction

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Phenolase exist in common daily activates since they are found on a variety of fruits and vegetables that are consumed. When the fruits and vegetables such as mushrooms, potatoes and bananas are damaged it tends to turn brown over time because the enzyme phenolase present in them are exposed to oxygen and hence oxidizes the substrate found in the cells. 

Enzyme browning reaction can be controlled by several procedures such as the inactivation of the enzyme and the removal of oxygen. A process called blanching is a very effective way in controlling enzyme browning reaction by heating the fruits or vegetable to denature the enzyme before storing. In addition to heating the fruit or vegetable can be stored in ice to prevent oxidation.  Also the addition of citric acid like those found in lime or vinegar decreases the pH at which the enzyme would normally function hence inhibiting enzymatic activity.

Test Your Knowledge ……. TCA….

quiz-night

1. Name the enzyme found in the Kreb Cycle (TCA) that is similar to pyruvate dehydrogenase.

(A) Alpha-ketoglutarate Dehydrogenase

(B) NADH Dehydrogenase

(C) Coenzyme A

(D) Malate Dehydrogenase

(E) Fumarase

 

What is the correct order os step 1 in the Kreb Cycle

1. S-citryl CoA undergoes hydrolysis to from citrate

2. An enzyme removes a proton from the CH3 in CoA

3. This causes a strong forward reaction.

4. The negatively charged CH2 bond to the oxaloacetate carbon

 

(A) 1, 2, 3, 4

(B) 2, 1, 3, 4

(C) 3,1,4, 2,

(D) 2, 4, 1, 3

(E) 4, 2, 3, 1

 

Ah Riddle Ah Riddle Ah Ree… ETC Quiz

quiz_w1

1. Why does ATP synthase undergo conformational changes in the electron transport chain?

1. To generate ATP from ADP + P

2. To move protons from the inter-membrane to the matrix of the mitochondria

3. To move protons from the inter-membrane to the outer membrane

4. To generate ADP + P using ATP

 

(A) 1 only

(B) 1, 2,

(C) 2, 3

(D) 2, 4

(E) All of the above

 

2. Rotenone is an inhibitor that disrupts the electron transport chain complexes. Which of the complex is disrupted?

(A) Complex 1

(B) Complex 2

(C) Complex 3

(D) Complex 4

(E) ATP Synthase Complex

 

Inhibitors of ETC

Cyanide poison inhibits complex 2, cytochrome oxidase, of the electron transport chain. It binds to the Fe+++ in the hem group in complex 2 interfering with the pumping of the protons and the flow of reaction. Therefore a electrochemical gradient would not be creating hence ATP synthase would not release the ATP for the ions to move into the matrix.

Rotenone acts much like cyanide, however it inhibits complex 1. NADP Dehydrogenase.

Oligomycin  inhibits mitochondrial H+-ATP synthase by binding to the Oligomycin sensitivity-conferring protein (OSCP) at the F(o) subunits 6 and 9  which are found in the stalk of the F1F0-ATPase complex. This binding blocks the proton conductance and inhibits the synthesis of mitochondrial ATP. Therefore the electrochemical gradient would increase in the intermembrane and decrease in the matrix until the protons runs out.

2, 4 DNP is known as an uncouple as it only affects one complex. It is hydrophobic and has the ability to release protons from the inter-membrane space to the matrix by perforating the wall on the inner membrane while it is lodge in it. Therefore no energy is require for the active transport and the energy made is released tot he surroundings as heat. The ETC still works.

TCA – Link Reaction

tcae

 SUMMARY OF THE LINK REACTION

  • Two pyruvates are formed from glycolysis in the cytoplasm.
  • Most of the energy that was stored in the glucose molecule is still present in pyruvic acid.
  • When oxygen is present, pyruvate is transported to the mitochondrial matrix and more reactions take place, freeing more energy.
  • The link reaction involves oxidative decarboxylation of pyruvate: oxidative because an H+ is removed; decarboxylation because a CO2 is removed.
  • And the link reaction involves the reduction of NAD+ to NADH, and it produces acetyl coenzyme A (acetyl CoA) and CO2.
  • Net yield of 2reduced NADH per glucose
  • Pyruvate Dehydrogenase Cofactors

    • TPP: thiazolium ring adds to  carbon of pyruvate, then stabilizes the resulting carbanion by acting as an electron sink.
    •  Lipoic acid: oxidizes pyruvate to level of acetate (acetylCoA), and activates acetate as a thioester.
    • CoA-SH: activates acetate as thioester.
    • FAD: oxidizes lipoic acid.
    • NAD+ : oxidizes FAD.