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Krebs cycle

2011-11-14T18:55:05Z

104097300:

==== Introduction ====

The Krebs Cycle can also be called the [[Citric Acid Cycle|Citric Acid Cycle]] (CAC) or the [[Tricarboxylic Acid|Tricarboxylic Acid]] (TCA) Cycle. This cycle takes place in the matrix of [[Mitochondria|mitochondria]] and is the first step of aerobic processing in the cell. The process [[Oxidation|oxidises]] glucose derivatives, [[Fatty acids|fatty acids]] and [[Amino acids|amino acids]] to [[Carbon dioxide|carbon dioxide]] (CO2) through a series of [[Enzyme|enzyme]] controlled steps. The purpose of the Krebs Cycle is to collect (eight) high-energy electrons from these fuels by oxidising them, which are transported by activated carriers [[NADH|NADH and]] [[FADH2|FADH2]] to the [[Electron transport chain|electron transport chain]]. The Krebs Cycle is also the source for the precursors of many other molecules and is therefore an amphibolic pathway (meaning it is both [[Anabolic|anabolic]] and [[Catabolic|catabolic]]) <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p475-476</ref><ref>Alberts, B. Johnson, A. Lewis, J. Raff, M. Roberts, K. and Walter, P. (2008). Molecular Biology of The Cell. 5th ed. New York and Abingdon: Garland Science. p97-98</ref>.

 [[Image:Pyruvate+CAC.jpg|center|280x220px|Pyruvate+CAC.jpg]] 

==== The 8 Steps of the Krebs Cycle ====

===== Step 0 =====

''* Not actually part of the Krebs Cycle. This step simply links glycolysis to the Krebs Cycle.'' Glycolysis produces [[Pyruvate|pyruvate]] which, under aerobic conditions, gets moved into the [[Mitochondria|mitochondria]] via a carrier protein within the membrane. There it is oxidatively decarboxylated by a huge enzyme complex called the [[Pyruvate Dehydrogenase Complex|pyruvate dehydrogenase complex]]. This reaction is irreversible and requires [[Coenzyme A|coenzyme A]] as well as producing 1 CO2 and picks up two electrons by [[NADH|NAD]][[NADH|+]]  <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p477</ref>.

{| width="70%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Pyruvate(3C) + CoA(1C) + NAD+
| [[Pyruvate Dehydrogenase Complex|pyruvate dehydrogenase]]
| Acetyl CoA(2C) + CO2 + NADH + H+
| Oxidative decarboxylation
| Irreversible
|}

 

===== Step 1 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Acetyl CoA (2C) + Oxaloacetate (4C) + H20
| Citrate synthase
| Citrate (6C) + CoA + H+
| Aldol [[Condensation Reaction|condensation and]] [[Hydrolysis|hydrolysis]]
| Irreversible
|}

The [[Oxaloacetate|oxaloacetate]] and [[Acetyl CoA|acetyl CoA]] are first condensed forming [[Citryl CoA|citryl CoA]] which is then hydrolysed to CoA and [[Citrate|citrate]]. This second step gives the power for the whole reaction to move forward and make [[Citrate|citrate]]. The [[Oxaloacetate|oxaloacetate]] binds to the [[Citrate synthase|citrate synthase]] enzyme first and causes a big conformational change which causes it to make the [[Acetyl CoA|acetyl CoA]] binding site by altering the enzyme from the open to the closed form. The enzyme performs the condensation by getting both substrates close together, positioning them appropriately and then polarising specific bonds. When [[Citryl CoA|citryl CoA]] is formed, it causes the enzyme’s active site to close fully <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p482-483</ref>.

===== Step 2 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Citrate (6C)
| Aconitase
| Isocitrate (6C)
| Reshuffling (dehydrating and then hydrating)
| Reversible
|}

This step is required to relocate the [[Hydroxyl group|hydroxyl group]] on the citrate molecule so that [[Oxidative decarboxylation|oxidative decarboxylation]] can take place. The enzyme aconitase contains iron that is not bonded to a heme which means it is an [[Iron-sulphur protein|iron-sulphur protein]] and this 4Fe-S cluster is what takes part in the dehydrating and hydrating of [[Citrate|citrate]] <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p484</ref>. 

===== Step 3 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Isocitrate (6C) + NAD+
| Isocitrate dehydrogenase
| α-Ketoglutarate (5C) + CO2 + NADH
| Oxidative decarboxylation
| Reversible
|}

[[Oxalosuccinate|Oxalosuccinate]] is an unstable [[Β-ketoacid|β-ketoacid]] and is the intermediate of the reaction in step 3. How quickly the [[Α-Ketoglutarate|α-Ketoglutarate]] is produced determines the rate of the whole cycle <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p484-485</ref>. 

===== Step 4 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| α-Ketoglutarate (5C) + NAD+ + CoA
| α-Ketoglutarate dehydrogenase complex
| Succinyl CoA (4C) + CO2 + NADH
| Oxidative decarboxylation
| Reversible
|}

The [[Α-ketoglutarate dehydrogenase|α-ketoglutarate dehydrogenase]] complex is a group of three kinds of enzymes and is homologous to the [[Pyruvate dehydrogenase|pyruvate dehydrogenase]] complex in [[Glycolysis|glycolysis]]. Their oxidative decarboxylation process is similar because [[Pyruvate|pyruvate]] and [[Α-ketoglutarate|α-ketoglutarate]] are both [[Α-ketoacid|α-ketoacid]] are the process has analogous reaction mechanisms <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p485</ref>. 

===== Step 5 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Succinyl CoA (4C) + Pi + GDP + H20
| Succinyl CoA synthase
| Succinate (4C) + GTP + CoA
| Substrate-level phosphorylation
| Reversible
|}

The [[Phosphorylation|phosphorylation]] of [[GDP|GDP]] to [[GTP|GTP]] is coupled by the breakdown of the [[Thioester bond|thioester bond]] in the molecule of [[Succinyl CoA|succinyl CoA]] which powers it.<ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p485-486</ref> 

===== Step 6 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Succinate (4C) + FAD
| Succinnate dehydrogenase
| Fumerate (4C) + FADH2
| Oxidation 
| Reversible
|}

This is where the regeneration of oxaloacetate, the last stage, in the Krebs cycle begins. The hydrogen acceptor in this step is [[FAD|FAD]] instead of [[NAD|NAD]] because the [[Free energy|free energy]] change, ΔG, due to the oxidation is only high enough to be able to reduce [[FAD|FAD]] and not enough to reduce [[NAD+|NAD]][[NAD+|+]]. [[Succinnate dehydrogenase|Succinnate dehydrogenase]] is also an [[Iron-sulphur protein|iron-sulphur protein]] just like aconitase but is imbedded in the inner membrane of the [[Mitochondria|mitochondria]] and is therefore the direct link between the Krebs cycle and the electron transport chain.  ===== ===== The [[FADH2|FADH]][[FADH2|2]] donates its two electrons to the Fe-S clusters in the enzyme which then transfers them onto [[Coenzyme Q|coenzyme Q]] (CoQ) that passes through the [[Electron transport chain|electron transport chain]] <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p486</ref>. 

===== Step 7  =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Fumerate (4C) + H20
| Fumerase
| L- Malate (4C)
| Hydration
| Reversible
|}

The reason why only the L form of the [[Malate|malate]] is formed is because he enzyme fumerase is stereospecific and only adds H+ and OH- to one side of the double bond in fumarate <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p487-488</ref>. 

===== Step 8 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| L-Malate (4C) + NAD+
| Malate dehydrogenase
| Oxaloacetate (4C) + NAD + H+
| Oxidation
| Reversible
|}

This oxidation reaction is moved along by the use of [[Oxaloacetate|oxaloacetate]] and [[NADH|NADH]] <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p488</ref><ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p489</ref>.

 

[[Image:CAC detail.jpg|center|600x500px|CAC detail.jpg]]

==== Products of the Krebs Cycle ====

Overall: Acetyl CoA + 3NAD+ + FAD + GDP + Pi + H20 gives -> 2CO2 + 3NADH + FADH2 + GTP + CoA + 2H+ (Remember: multiple these by 2 because for every 1 [[Glucose|glucose]] we get 2 [[Pyruvate|pyruvate]] [[Molecules|molecules]] and thus 2 [[Acetyl CoA|acetyl CoA]] molecules) <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p488</ref>

==== Control of the Krebs Cycle ====

The Krebs Cycle is inhibited by high energy levels and encouraged by low energy levels in the cell. There are 3 control points in the Krebs Cycle:

#[[Pyruvate dehydrogenase|Pyruvate dehydrogenase]]
#[[Isocitrate dehydrogenase|Isocitrate dehydrogenase]]
#[[Α-Ketoglutarate dehydrogenase|α-Ketoglutarate dehydrogenase]] 

 1. '''Pyruvate dehydrogenase''' [[Image:Control of CAC.jpg|right|500x400px|Control_of_CAC.jpg]]

Encouraged by high levels of: [[ADP|ADP]] and [[Pyruvate|pyruvate]]

Inhibited by high levels of: [[ATP|ATP]], [[Acetyl CoA|acetyl CoA]] and [[NADH|NADH]]

''[[Acetyl CoA|Acetyl CoA]] inhibits the transacetylase component, E2, by binding to it directly. NADH on the other hand inhibits the dihydrolipoyl dehydrogenase E3. The way in which this enzyme complex is regulated is through covalent modification by phosphorylation. To switch off the activity of the enzyme a specific kinase will phosphorylate the enzyme complex and to reactivate it a specific phophotase is needed.'' 

 

2. '''Isocitrate dehydrogenase''' Inhibited by high levels of: [[ATP|ATP]] and [[NADH|NADH]] Encouraged by high levels of: [[ADP|ADP]] ADP allosterically encourages the enzyme by increasing its affinity for isocitrate. 

 

3. '''α-Ketoglutarate dehydrogenase''' Inhibited by high levels of: [[ATP|ATP]], [[Succinyl CoA|succinyl CoA]] and [[NADH|NADH]] <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p490-493</ref>

 

 

 

==== Anabolic Properties of the Krebs Cycle ====

[[Citrate|Citrate]] -> sterols and fatty acids [[Α-Ketoglutarate|α-Ketoglutarate]] -> [[Glutamate|glutamate]], other amino acids and purines [[Succinyl CoA|Succinyl CoA]] -> porphyrins, heme and chlorophyll [[Oxaloacetate|Oxaloacetate]] -> aspartate, other amino acids, purines and pyrimidines <ref>Alberts, B. Johnson, A. Lewis, J. Raff, M. Roberts, K. and Walter, P. (2008). Molecular Biology of The Cell. 5th ed. New York and Abingdon: Garland Science. p99</ref>

[[Image:Anabolic properties of CAC.jpg|left|400x300px|Anabolic_properties_of_CAC.jpg]]

 

 

 

 

 

 

 

 

 

 

 

==== References ====

<references />

Krebs cycle

2011-11-14T18:54:10Z

104097300:

Krebs cycle

2011-11-14T18:51:21Z

104097300:

==== Introduction ====

The Krebs Cycle can also be called the [[Citric Acid Cycle|Citric Acid Cycle]] (CAC) or the [[Tricarboxylic Acid|Tricarboxylic Acid]] (TCA) Cycle. This cycle takes place in the matrix of [[Mitochondria|mitochondria]] and is the first step of aerobic processing in the cell. The process [[Oxidation|oxidises]] glucose derivatives, [[Fatty acids|fatty acids]] and [[Amino acids|amino acids]] to [[Carbon dioxide|carbon dioxide]] (CO2) through a series of [[Enzyme|enzyme]] controlled steps. The purpose of the Krebs Cycle is to collect (eight) high-energy electrons from these fuels by oxidising them, which are transported by activated carriers [[NADH|NADH and]] [[FADH2|FADH2]] to the [[Electron transport chain|electron transport chain]]. The Krebs Cycle is also the source for the precursors of many other molecules and is therefore an amphibolic pathway (meaning it is both [[Anabolic|anabolic]] and [[Catabolic|catabolic]]) <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p475-476</ref><ref>Alberts, B. Johnson, A. Lewis, J. Raff, M. Roberts, K. and Walter, P. (2008). Molecular Biology of The Cell. 5th ed. New York and Abingdon: Garland Science. p97-98</ref>.

 [[Image:Pyruvate+CAC.jpg|center|280x220px|Pyruvate+CAC.jpg]] 

==== The 8 Steps of the Krebs Cycle ====

===== Step 0 =====

''* Not actually part of the Krebs Cycle. This step simply links glycolysis to the Krebs Cycle.'' Glycolysis produces [[Pyruvate|pyruvate]] which, under aerobic conditions, gets moved into the [[Mitochondria|mitochondria]] via a carrier protein within the membrane. There it is oxidatively decarboxylated by a huge enzyme complex called the [[Pyruvate Dehydrogenase Complex|pyruvate dehydrogenase complex]]. This reaction is irreversible and requires [[Coenzyme A|coenzyme A]] as well as producing 1 CO2 and picks up two electrons by [[NADH|NAD]][[NADH|+]]  <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p477</ref>.

{| width="70%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Pyruvate(3C) + CoA(1C) + NAD+
| [[Pyruvate Dehydrogenase Complex|pyruvate dehydrogenase]]
| Acetyl CoA(2C) + CO2 + NADH + H+
| Oxidative decarboxylation
| Irreversible
|}

 

===== Step 1 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Acetyl CoA (2C) + Oxaloacetate (4C) + H20
| Citrate synthase
| Citrate (6C) + CoA + H+
| Aldol [[Condensation Reaction|condensation and]] [[Hydrolysis|hydrolysis]]
| Irreversible
|}

The [[Oxaloacetate|oxaloacetate]] and [[Acetyl CoA|acetyl CoA]] are first condensed forming [[Citryl CoA|citryl CoA]] which is then hydrolysed to CoA and [[Citrate|citrate]]. This second step gives the power for the whole reaction to move forward and make [[Citrate|citrate]]. The [[Oxaloacetate|oxaloacetate]] binds to the [[Citrate synthase|citrate synthase]] enzyme first and causes a big conformational change which causes it to make the [[Acetyl CoA|acetyl CoA]] binding site by altering the enzyme from the open to the closed form. The enzyme performs the condensation by getting both substrates close together, positioning them appropriately and then polarising specific bonds. When [[Citryl CoA|citryl CoA]] is formed, it causes the enzyme’s active site to close fully <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p482-483</ref>.

===== Step 2 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Citrate (6C)
| Aconitase
| Isocitrate (6C)
| Reshuffling (dehydrating and then hydrating)
| Reversible
|}

This step is required to relocate the [[Hydroxyl group|hydroxyl group]] on the citrate molecule so that [[Oxidative decarboxylation|oxidative decarboxylation]] can take place. The enzyme aconitase contains iron that is not bonded to a heme which means it is an [[Iron-sulphur protein|iron-sulphur protein]] and this 4Fe-S cluster is what takes part in the dehydrating and hydrating of [[Citrate|citrate]] <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p484</ref>. 

===== Step 3 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Isocitrate (6C) + NAD+
| Isocitrate dehydrogenase
| α-Ketoglutarate (5C) + CO2 + NADH
| Oxidative decarboxylation
| Reversible
|}

[[Oxalosuccinate|Oxalosuccinate]] is an unstable [[Β-ketoacid|β-ketoacid]] and is the intermediate of the reaction in step 3. How quickly the [[Α-Ketoglutarate|α-Ketoglutarate]] is produced determines the rate of the whole cycle <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p484-485</ref>. 

===== Step 4 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| α-Ketoglutarate (5C) + NAD+ + CoA
| α-Ketoglutarate dehydrogenase complex
| Succinyl CoA (4C) + CO2 + NADH
| Oxidative decarboxylation
| Reversible
|}

The [[Α-ketoglutarate dehydrogenase|α-ketoglutarate dehydrogenase]] complex is a group of three kinds of enzymes and is homologous to the [[Pyruvate dehydrogenase|pyruvate dehydrogenase]] complex in [[Glycolysis|glycolysis]]. Their oxidative decarboxylation process is similar because [[Pyruvate|pyruvate]] and [[Α-ketoglutarate|α-ketoglutarate]] are both [[Α-ketoacid|α-ketoacid]] are the process has analogous reaction mechanisms <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p485</ref>. 

===== Step 5 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Succinyl CoA (4C) + Pi + GDP + H20
| Succinyl CoA synthase
| Succinate (4C) + GTP + CoA
| Substrate-level phosphorylation
| Reversible
|}

The [[Phosphorylation|phosphorylation]] of [[GDP|GDP]] to [[GTP|GTP]] is coupled by the breakdown of the [[Thioester bond|thioester bond]] in the molecule of [[Succinyl CoA|succinyl CoA]] which powers it.<ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p485-486</ref> 

===== Step 6 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Succinate (4C) + FAD
| Succinnate dehydrogenase
| Fumerate (4C) + FADH2
| Oxidation 
| Reversible
|}

This is where the regeneration of oxaloacetate, the last stage, in the Krebs cycle begins. The hydrogen acceptor in this step is [[FAD|FAD]] instead of [[NAD|NAD]] because the [[Free energy|free energy]] change, ΔG, due to the oxidation is only high enough to be able to reduce [[FAD|FAD]] and not enough to reduce [[NAD+|NAD]][[NAD+|+]]. [[Succinnate dehydrogenase|Succinnate dehydrogenase]] is also an [[Iron-sulphur protein|iron-sulphur protein]] just like aconitase but is imbedded in the inner membrane of the [[Mitochondria|mitochondria]] and is therefore the direct link between the Krebs cycle and the electron transport chain.  ===== ===== The [[FADH2|FADH]][[FADH2|2]] donates its two electrons to the Fe-S clusters in the enzyme which then transfers them onto [[Coenzyme Q|coenzyme Q]] (CoQ) that passes through the [[Electron transport chain|electron transport chain]] <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p486</ref>. 

===== Step 7  =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| Fumerate (4C) + H20
| Fumerase
| L- Malate (4C)
| Hydration
| Reversible
|}

The reason why only the L form of the [[Malate|malate]] is formed is because he enzyme fumerase is stereospecific and only adds H+ and OH- to one side of the double bond in fumarate <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p487-488</ref>. 

===== Step 8 =====

{| width="80%" border="1" cellpadding="1" cellspacing="1"
|-
| Reactants
| Enzyme
| Products
| Reaction type
| (Ir)reversible
|-
| L-Malate (4C) + NAD+
| Malate dehydrogenase
| Oxaloacetate (4C) + NAD + H+
| Oxidation
| Reversible
|}

This oxidation reaction is moved along by the use of [[Oxaloacetate|oxaloacetate]] and [[NADH|NADH]] <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p488</ref><ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p489</ref>.

 

===== [[Image:CAC detail.jpg|center|600x500px|CAC detail.jpg]] =====

==== Products of the Krebs Cycle ====

Overall: Acetyl CoA + 3NAD+ + FAD + GDP + Pi + H20 gives -> 2CO2 + 3NADH + FADH2 + GTP + CoA + 2H+ (Remember: multiple these by 2 because for every 1 [[Glucose|glucose]] we get 2 [[Pyruvate|pyruvate]] [[Molecules|molecules]] and thus 2 [[Acetyl CoA|acetyl CoA]] molecules) <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p488</ref>

==== Control of the Krebs Cycle ====

The Krebs Cycle is inhibited by high energy levels and encouraged by low energy levels in the cell. There are 3 control points in the Krebs Cycle:

#[[Pyruvate dehydrogenase|Pyruvate dehydrogenase]]
#[[Isocitrate dehydrogenase|Isocitrate dehydrogenase]]
#[[Α-Ketoglutarate dehydrogenase|α-Ketoglutarate dehydrogenase]] 

 1. '''Pyruvate dehydrogenase''' [[Image:Control of CAC.jpg|right|500x400px|Control_of_CAC.jpg]]

Encouraged by high levels of: [[ADP|ADP]] and [[Pyruvate|pyruvate]]

Inhibited by high levels of: [[ATP|ATP]], [[Acetyl CoA|acetyl CoA]] and [[NADH|NADH]]

''[[Acetyl CoA|Acetyl CoA]] inhibits the transacetylase component, E2, by binding to it directly. NADH on the other hand inhibits the dihydrolipoyl dehydrogenase E3. The way in which this enzyme complex is regulated is through covalent modification by phosphorylation. To switch off the activity of the enzyme a specific kinase will phosphorylate the enzyme complex and to reactivate it a specific phophotase is needed.'' 

 

2. '''Isocitrate dehydrogenase''' Inhibited by high levels of: [[ATP|ATP]] and [[NADH|NADH]] Encouraged by high levels of: [[ADP|ADP]] ADP allosterically encourages the enzyme by increasing its affinity for isocitrate. 

 

3. '''α-Ketoglutarate dehydrogenase''' Inhibited by high levels of: [[ATP|ATP]], [[Succinyl CoA|succinyl CoA]] and [[NADH|NADH]] <ref>Berg J.M, Tymoczko J.L, Stryer, L (2007). Biochemistry. 6th ed. New York: W. H. Freeman and Company. p490-493</ref>

===== =====

 

===== =====

==== Anabolic Properties of the Krebs Cycle ====

[[Citrate|Citrate]] -> sterols and fatty acids [[Α-Ketoglutarate|α-Ketoglutarate]] -> [[Glutamate|glutamate]], other amino acids and purines [[Succinyl CoA|Succinyl CoA]] -> porphyrins, heme and chlorophyll [[Oxaloacetate|Oxaloacetate]] -> aspartate, other amino acids, purines and pyrimidines <ref>Alberts, B. Johnson, A. Lewis, J. Raff, M. Roberts, K. and Walter, P. (2008). Molecular Biology of The Cell. 5th ed. New York and Abingdon: Garland Science. p99</ref>

===== [[Image:Anabolic properties of CAC.jpg|left|400x300px|Anabolic_properties_of_CAC.jpg]] =====

===== =====

===== =====

==== References ====

<references />

File:Pyruvate+CAC.jpg

2011-11-14T18:33:00Z

104097300: Step 0 and Cycle as Steps 1-8

Step 0 and Cycle as Steps 1-8

File:Control of CAC.jpg

2011-11-14T18:32:16Z

104097300: Control of the Krebs cycle by catalysis of irreversible steps

Control of the Krebs cycle by catalysis of irreversible steps

File:CAC detail.jpg

2011-11-14T18:30:51Z

104097300: Krebs cycle in detail

Krebs cycle in detail

File:Anabolic properties of CAC.jpg

2011-11-14T18:30:09Z

104097300: Anabolic properties of the Krebs Cycle

Anabolic properties of the Krebs Cycle

Enac

2011-11-14T18:21:01Z

104097300:

=== Introduction ===

ENaC, also called the amiloride-sensitive sodium channel (ASC), is an epithelial [[Sodium|Na+]] channel found on the apical side of polar epithelial cells of the [[Kidney|kidney]], [[Colon|colon]], [[Lung|lung]] and sweat glands. It is a non-voltage-sensitive [[Ions|ion]] channel permeable to [[Sodium|Na+]] [[Ions|ions]]<ref>http://prosite.expasy.org/PDOC00926#ref4</ref>. The Na+ ions move from the lumen to the [[Blood|blood side]] of epithelial [[Cell|cells]], ie. they are reabsorbed. In the kidney, ENaC is located on [[Principal cell|principal cells]] in the [[Distal tubule|distal convoluted tubule]] and [[Collecting duct|collecting duct]] where its role is to retain Na+. In the colon, the ENaC reabsorbs Na+ from the diet in the lumen and contributes to the body’s overall Na+ balance. In the lungs, ENaC is vital for neonates where it reabsorbs and removes the amniotic fluid that fills the interior of the lungs allowing them to fill with air for the first breath. 

 

=== Structure ===

The channel is trimeric, ie. it is made of three [[Homology|homologous subunits]] called α, β and γ, all which must be co-expressed for the channel to be able to work.  [[Image:Structure 2.jpg|right|190x182px|Structure 2.jpg]]

[[Image:Structure 1.jpg|left|273x186px|Structure 1.jpg]] 

 

''<- The first few points of the curve on the far right-hand side, are the experiment with each of the three subunits separately. Then moving onto combinations of two subunits and finally all three which gives the best response''.<ref>Canessa et al Nature 367, 3rd Feb, 1994</ref> 

 

 

 

 

 [[Image:Structure 3.jpg|left|153x170px|Structure 3.jpg]] The ring in between subunits α, β and γ determines the selectivity of the channel.

[[Amino acids|Amino acids]] 587 to 589 ([[Glycine|glycine]] to [[Serine|serine]]) make up the selectivity filter.

Amino acid at position 583 (serine) is the amiloride [[Enzyme active site|binding site]].

<ref>Stockland JD et al, Life, 60(9): 620–628</ref>  [[Image:Structure 4.jpg|right|245x367px|Structure 4.jpg]]

 

 

 

 

The channel has an intracellular N-terminus in the [[Cytoplasm|cytoplasm]] that is followed by the first [[Transmembrane|transmembrane domain]] which extends into a large extracellular loop and goes back into the membrane as the second transmembrane domain and ends at a C-terminal intracellular tail in the cytoplasm.

 [[Image:Structure 5.jpg|left|243x159px|Structure 5.jpg]]

 

 

The large extracellular loop has [[Cysteine|cysteine]] rich domains (CRDs) that help regulate the channel.

 <ref>Pflugers Arch. 2010 June ; 460(1): 1–17. doi:10.1007/s00424-010-0827-z</ref> 

=== Regulation of Na+ Absorption ===

==== Short term ====

'''1''' – ENaC and Na+ entry is the rate limiting step of Na+ absorption 

[[Image:Regulation 1.jpg|left|271x188px|Regulation 1.jpg]]

 

 

An increase in the external Na+ means there is a direct increase in the Na+ moving into the cell but only to a certain extent after which ENaC intrinsically down regulates and inhibits itself using its [[Tertiary Protein Structure|tertiary structure]], leading to a decrease in the [[Open state probability|open state probability]] (O.S.P) and the Na+ influx becomes steady.

 

 

 

 

 

 '''2 '''– Activation of ENaC by proteolitic cleavage [[Image:Regulation 2.jpg|right|478x203px|Regulation 2.jpg]]

 

*ENaC moves to the membrane in its inactive form. The first step to activation involves a [[Protease|protease furin]] to cleave a 26mer with a 8mer inhibitory tract from the α subunit. This makes ENaC partially activated. 
*Another protease, usually [[Prostatin|prostatin]], cleaves a 43mer [[Polypeptide|peptide from]] the γ subunit of ENaC fully activating the channel.

  <ref>Soundararajan R et al. J. Biol. Chem. 2010;285:30363-30369</ref>

 

 

==== Long Term ====

'''1''' – In the colon, late distal tubule and collecting duct when Na+ absorption increases, K+ secretion increases as a result due to the lumen being more [[Electronegativity|electronegative]] than the blood basolateral side of the cells.

 '''2''' – Hormone Control The steroid [[Hormones|hormone]] [[Aldosterone|aldosterone increases]] the insertion of ENaCs into the membrane and their open state probability but usually not the channel’s synthesis unless in the colon. When [[Blood pressure|blood pressure]] and volume in the body are low, the [[Renin-Angiotensin System|RAAS system]] will be activated by Renin release from the juxtaglomerular cells in the kidney in response to low afferent tension and Na+ flow. As this eventually results in a production of aldosterone, ENaC activity will increase and more Na+ will be retained so [[Osmolarity|osmolarity]] and volume increase, resulting in an increase in blood pressure that returns it back to a normal range. [[Image:Regulation 3 (RAAS).jpg|center|134x143px|Regulation 3 (RAAS).jpg]]

From the bloodstream, aldosterone crosses the cell membrane and binds its corticosteroid [[Receptor|receptor]] found in the cytoplasm. The two travel to the [[Nucleus|nucleus]] where they act as a [[Transcription|transcription factor]] and increase the transcription of [[MRNA|mRNA that]] encodes aldosterone induced/regulated proteins ([[Aldosterone induced proteins|AIT/ARTs]]). These proteins increase cell surface ENaC and Na+/K+ ATP-ase density.

[[Image:Regulation 4.jpg|center|239x195px|Regulation 4.jpg]] 

One way this is done is by up regulating serum and glucocorticoid regulated kinase ([[SGK|SGK]]) which is the first protein translated from mRNA. SGK phosphorylates a [[Serine|serine]] on [[Nedd4|Nedd4]] which disables it from marking ENaC for degradation and thus the channel stays on the membrane. Nedd4 is a ubiquitin ligase which marks ENaC for degradation by binding to the C terminal of the channel that is rich in [[Proline|proline]]. When bound, Nedd4 will ligate [[Ubiquitin|Ubiquitin]] to ENaC’s N terminus which marks the channel for retrieval. [[Image:Regulation 5.jpg|center|239x200px|Regulation 5.jpg]] 

=== Disease and Treatment ===

The [[Gene|gene encoding]] for ENaC is found on [[Chromosome|chromosome ]]4 at map 4q31.3-q32.<ref>NP_059115.1</ref> [[Mutation|Mutations in]] the genes encoding the cytoplasmic C-terminal of either the β or γ subunit will result in [[Liddle Syndrome|Liddle’s Syndrome]]. The faulty C-terminal of ENaC means that [[Nedd4|Nedd4]] is unable to bind to it and cannot ligate Ubiquitin so the channel is not marked for retrieval and ENaC activity stays high in the cell. The condition results in [[Hypertension|hypertension]], [[Hypokalemia|hypokalemia]] and sometimes [[Alkalosis|alkalosis]]. This is because too much Na+ is retained, elevating blood volume and thus pressure (hypertension) which suppresses the [[Renin-Angiotensin System|RAAS system]]. The elevated Na+ means that more K+ is also secreted, leaving the blood with low K+ levels (hypokalemia). The blood is also more electropositive than normal so H+ ions sometimes expelled from the cell via the [[Apical membrane|apical side]] leaving the blood alkaline (alkalosis).

Treatment for [[Liddle Syndrome|Liddle’s Syndrome]] comes in K+-sparing [[Diuretic|diuretics that]] act on the late distal tubule and collecting duct. Common drugs that fall into this category are [[Amiloride|amiloride]] or [[Triamterene|triamterene]]. Amiloride is a [[Cation|cationic]] drug at physiological state and acts as a high [[Affinity|affinity]] physical blocker to the channel by binding amino acid position 583. The drug is orally absorbed (15-25%) and has a [[Half life|half life]] of 21 hours. By blocking the channel is decreases Na+ retention and creates a more electropositive lumen thus reduce K+ and H+ secretion into it which makes the drug “K+-sparing”.

[[Image:Disease and treatment 1-amiloride from pubchem CID 2016231.jpg|center|357x143px|Disease and treatment 1-amiloride from pubchem CID 2016231.jpg]]<ref>PubChem CID 16231</ref> 

ENaC inhibition in the lungs can be useful for the treatment of [[Cystic fibrosis|cystic fibrosis]]. In normal individuals [[CFTR|CFTR]] inhibits ENaC and controls Na+ absorption but in [[Cystic fibrosis|CF]] patients, there is either no [[CFTR|CFTR]] or it is faulty, which results in no inhibition of ENaC and thus too much Na+ being reabsorbed and decrease in airway surface liquid ([[Airway surface liquid|ASL]]) . A potential [[Pharmacotherapy of Cystic Fibrosis|treatment to]] avoid this is to block ENaC with amiloride-like drugs such as [[Pharmacotherapy of Cystic Fibrosis|GS9411]].<ref>Nat. Med. May 2004. 10:452-453</ref>

[[Image:Disease and treatment 2.jpg|center|192x152px|Disease and treatment 2.jpg]] 

=== References ===

<references />

2011-11-14T00:02:01Z

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'''Hypokalemia '''is when the [[Blood|blood contains]] lower levels of potassium (K+) than normal. 

 

Some possible causes of hypokalemia may be:

*Antibiotics 
*Diarrhea 
*Diseases that affect the [[Kidney|kidneys]]' ability to retain potassium ([[Liddle Syndrome|Liddle syndrome]] and [[Cushing sydrome|Cushing syndrome]])
*[[Diuretic|Diuretics]]
*Eating disorders
*Eating large amounts of licorice or other foods with glycyrrhetinic acid 
*[[Magnesium|Magnesium deficiency]]
*Sweating
*Vomiting<ref>http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001510/</ref>

 

Symptoms of severe hypokalemia include:

*Abnormal heart rhythms
*Constipation
*Fatigue
*[[Muscle|Muscle damage]] 
*[[Muscle|Muscle weakness]] or spasms
*Paralysis (which can include the lungs)<ref>Mount DB, Zandi-Nejad K. Disorders of potassium balance. In: Brenner BM, ed. Brenner and Rector's The Kidney. 8th ed. Philadelphia, Pa: Saunders Elsevier; 2008:chap 15.</ref>

 

'''References'''

<references />

2011-11-13T21:17:57Z

104097300:

=== Introduction ===

ENaC, also called the amiloride-sensitive sodium channel (ASC), is an epithelial Na+ channel found on the apical side of polar epithelial cells of the kidney, colon, lung and sweat glands. It is a non-voltage-sensitive ion channel permeable to Na+ ions<ref>http://prosite.expasy.org/PDOC00926#ref4</ref>. The Na+ ions move from the lumen to the blood side of epithelial cells, ie. they are reabsorbed. In the kidney, ENaC is located on principal cells in the distal convoluted tubule and collecting duct where its role is to retain Na+. In the colon, the ENaC reabsorbs Na+ from the diet in the lumen and contributes to the body’s overall Na+ balance. In the lungs, ENaC is vital for neonates where it reabsorbs and removes the amniotic fluid that fills the interior of the lungs allowing them to fill with air for the first breath. 

=== Structure ===

The channel is trimeric, ie. it is made of three homologous subunits called α, β and γ, all which must be co-expressed for the channel to be able to work<ref>Canessa et al Nature 367, 3rd Feb, 1994</ref>. 

 [GRAPH: structure 1]

The ring in between subunits α, β and γ determines the selectivity of the channel. Amino acids 587 to 589 (glycine to serine) make up the selectivity filter. Amino acid at position 583 (serine) is the amiloride binding site.

[DIAGRAM: structure 2]<ref>Stockland JD et al, Life, 60(9): 620–628</ref> [DIAGRAM: structure 3]

The channel has an intracellular N-terminus in the cytoplasm that is followed by the first transmembrane domain which extends into a large extracellular loop and goes back into the membrane as the second transmembrane domain and ends at a C-terminal intracellular tail in the cytoplasm.

[DIAGRAM: structure 4]<ref>http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2955882/</ref> [DIAGRAM: structure 5]

The large extracellular loop has cysteine rich domains (CRDs) that help regulate the channel.

=== Regulation of Na+ Absorption ===

==== Short term ====

'''1''' – ENaC and Na+ entry is the rate limiting step of Na+ absorption An increase in the external Na+ means there is a direct increase in the Na+ moving into the cell but only to a certain extent after which ENaC intrinsically down regulates and inhibits itself using its tertiary structure, leading to a decrease in the open state probability (O.S.P) and the Na+ influx becomes steady.

[GRAPH: Regulation 1]

 

'''2 '''– Activation of ENaC by proteolitic cleavage a) ENaC moves to the membrane in its inactive form. The first step to activation involves a protease furin to cleave a 26mer with a 8mer inhibitory tract from the α subunit. This makes ENaC partially activated. b) Another protease, usually prostatin, cleaves a 43mer peptide from the γ subunit of ENaC fully activating the channel. [DIAGRAM: Regulation 2]<ref>Soundararajan R et al. J. Biol. Chem. 2010;285:30363-30369</ref>

==== Long Term ====

'''1''' – In the colon, late distal tubule and collecting duct when Na+ absorption increases, K+ secretion increases as a result due to the lumen being more electronegative than the blood basolateral side of the cells.

 '''2''' – Hormone Control The steroid hormone aldosterone increases the insertion of ENaCs into the membrane and their open state probability but usually not the channel’s synthesis unless in the colon. When blood pressure and volume in the body are low, the RAAS system will be activated by Renin release from the juxtaglomerular cells in the kidney in response to low afferent tension and Na+ flow. As this eventually results in a production of aldosterone, ENaC activity will increase and more Na+ will be retained so osmolarity and volume increase, resulting in an increase in blood pressure that returns it back to a normal range. [Flow chart: Regulation 3]

From the bloodstream, aldosterone crosses the cell membrane and binds its corticosteroid receptor found in the cytoplasm. The two travel to the nucleus where they act as a transcription factor and increase the transcription of mRNA that encodes aldosterone induced/regulated proteins (AIT/ARTs). These proteins increase cell surface ENaC and Na+/K+ ATP-ase density.

[DIAGRAM: Regulation 4]

One way this is done is by up regulating serum and glucocorticoid regulated kinase (SGK) which is the first protein translated from mRNA. SGK phosphorylates a serine on Nedd4 which disables it from marking ENaC for degradation and thus the channel stays on the membrane. Nedd4 is a ubiquitin ligase which marks ENaC for degradation by binding to the C terminal of the channel that is rich in proline. When bound, Nedd4 will ligate Ubiquitin to ENaC’s N terminus which marks the channel for retrieval. [DIAGRAM: Regulation 5]

=== Disease and Treatment ===

 The gene encoding for ENaC is found on chromosome 4 at map 4q31.3-q32. Mutations in the genes encoding the cytoplasmic C-terminal of either the β or γ subunit will result in Liddle’s Syndrome. The faulty C-terminal of ENaC means that Nedd4 is unable to bind to it and cannot ligate Ubiquitin so the channel is not marked for retrieval and ENaC activity stays high in the cell. The condition results in hypertension, hypokalemia and sometimes alkalosis. This is because too much Na+ is retained, elevating blood volume and thus pressure (hypertension) which suppresses the RAAS system. The elevated Na+ means that more K+ is also secreted, leaving the blood with low K+ levels (hypokalemia). The blood is also more electropositive than normal so H+ ions sometimes expelled from the cell via the apical side leaving the blood alkaline (alkalosis).

Treatment for Liddle’s Syndrome comes in K+-sparing diuretics that act on the late distal tubule and collecting duct. Common drugs that fall into this category are amiloride or triamterene. Amiloride is a cationic drug at physiological state and acts as a high affinity physical blocker to the channel by binding amino acid position 583. The drug is orally absorbed (15-25%) and has a half life of 21 hours. By blocking the channel is decreases Na+ retention and creates a more electropositive lumen thus reduce K+ and H+ secretion into it which makes the drug “K+-sparing”.

<DIAGRAM: Disease 1>

ENaC inhibition in the lungs can be useful for the treatment of cystic fibrosis. In normal individuals CFTR inhibits ENaC and controls Na+ absorption but in CF patients, there is either no CFTR or it is faulty, which results in no inhibition of ENaC and thus too much Na+ being reabsorbed and decrease in airway surface liquid (AL) . A potential treatment to avoid this is to block ENaC with amiloride-like drugs such as GS9411. (Nat. Med. May 2004. 10:452-453)

<DIAGRAM: Disease 2>