TCA+Cycle


 * 28 August 2006**
 * TCA Cycle**
 * Dr. Maurice Manning, Ph.D.**

toc

=**TCA Cycle Overview**=


 * Tricarboxyclic Acid Cycle
 * Takes Acetic Acid and 2 H2O to break that down into 2 CO2 and 4 H2
 * H2 is used to generate ATP
 * Fully oxidize acetic acid
 * Carbohydrates, proteins and lipids all feed in to make Acetyl-CoA
 * Takes place in the mitochondria
 * Requires oxygen to be the ultimate receptor of electrons from hydrogen
 * In total, 3 NADH+H+, 1 FADH2+ and 1 ATP is made per turn
 * 3 NADH+H+, 1 FADH2+ go on to oxidative phosphorylation
 * NADH+H+ generate 2.5 ATP
 * FADH2+ generates 1.5 ATP
 * In total, 8 reactions
 * Citrate Synthase
 * Aconitase
 * Isocitrate Dehydrogenase
 * Α-ketoglutarate Dehydrogenase
 * Succinyl-CoA Synthetase
 * Succinate Dehydrogenase
 * Fumarase
 * Malate Dehydrogenase
 * 10 intermediates
 * Acetyl CoA (C2)
 * Oxaloacetate (C4)
 * Citrate (C6)
 * Cis-Aconitate (C6)
 * Isocitrate (C6)
 * Α-Ketoglutarate (C5)
 * Succinyl-CoA (C4)
 * Succinate (C4)
 * Fumarate (C4)
 * Malate (C4)

=**TCA Cycle in Schematic**=



=**TCA Cycle in Detail**=

**Reaction 1**

 * Acetyl CoA + Oxaloacetate --> Citrate
 * Uses 1 H2O and lose 1 CoA-SH
 * Catalyzed by citrate Synthase

**Reaction 2**

 * Citrate --> cis-Aconitate
 * H2O is lost
 * Catalyzed by Aconitase
 * Occurs as an equilibrium reaction
 * Citrate can be used to make fat
 * Citrate is a pro-chiral molecule
 * Although citrate is symmetrical, it reacts asymmetrically with aconitase
 * Cis-Aconitate --> isocitrate
 * H2O is reclaimed and the –OH is moved to the β-carbon
 * Catalyzed by Aconitase
 * Occurs in an equilibrium reaction

**Reaction 3**

 * Isocitrate --> α-ketoglutarate
 * First CO2 is lost, first NADH+H+ is generated
 * Catalyzed by Isocitrate dehydrogenase
 * This is the committed step of the TCA cycle
 * Decarboxylation and Dehydrogenation occur in this step
 * Strongly inhibited by the negative modulators NADH+H+ and ATP
 * Stimulated by ADP

**Reaction 4**

 * α-ketoglutarate --> Succinyl-CoA
 * Second CO2 is lost, second NADH+H+ is generated
 * Analogous to pyruvate dehydration reaction
 * Requires thyamaine, lipoic acid, co-enzyme A, FAD+, and NAD+
 * Catalyzed by α-ketoglutarate dehydrogenase
 * Succinyl-CoA is a high-energy thio-ester
 * Dehydrogenation step

**Reaction 5**

 * Succinyl-CoA --> Succinate
 * Phosphorylation of GDP to GTP by **substrate-level phosphorylation**
 * GTP later converted to ATP by nucleoside diphosphate kinase
 * Catalyzed by Succinyl CoA synthase
 * Succinyl-CoA is a precursor for heme
 * Odd-numbered fatty acids will oxidize to a C3 fatty acid which can become succinyl-CoA for further metabolism

**Reaction 6**

 * Succinate --> Fumarate
 * FADH2 is generated
 * Hydrogens removed in a trans fashion
 * Catalyzed by succinate dehydrogenase
 * Dehydrogenation step
 * This as an equilibrium step

**Reaction 7**

 * Fumarate --> Malate
 * H2O is lost
 * Catalyzed by fumarase

**Reaction 8**

 * Malate --> Oxaloacetate
 * The third NADH+H+ is generated
 * Catalyzed by malate dehydrogenase
 * Dehydrogenation step

**Overall Reaction**
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O --> 2 CO2 + 3 NADH+H+ + FADH2 + GTP

=**Acetyl CoA Formation**=


 * Formed from pyruvate from glucose or from fatty acids by β-oxidation
 * Pyruvate + NAD+ + CoA --> Acetyl-CoA + NADH+H+ + CO2
 * Catalyzed using Pyruvate Dehydrogenase Complex (PDC)
 * Involves thyamaine paraphosphate, lipoic acid, co-enzyme A, FAD+, and NAD+
 * Reaction is reversible in animal tissues
 * Obligatory step for the entry of all carbohydrates in the TCA cycle
 * Reaction requires multienzyme PDC of 3 different enzymes, 5 different co-enzymes, plus 2 enzymes which regulate PDH
 * Enzymes required are:
 * Pyryvate Dehydrogenase (regulatory enzyme)
 * Dihydrolipoyltransacetylase
 * Diydrolipoyl’ dehydrogenase
 * Five required co-enzymes are:
 * Thiamine paraphosphate
 * Lipoic Acid
 * Co-enzyme A
 * FAD
 * NAD+
 * Pyruvate + Co-Enzyme A --> Aceytl CoA + NADH+H+, CO2

**Regulation of Pyruvate Dehydrogenase**

 * Covalent Modification
 * Active PDH is unphosphorylated
 * Ca2+ activates PDH phosphatase
 * Inactive PDH is phosphorylated
 * ATP acts as a substrate for PDH kinase
 * Both phosphatase and kinase are part of PDC
 * Byproduct inhibition
 * Acetyl-CoA and NADH+H+ inhibits PDH
 * Acceptor control
 * Adequate amounts of NAD+ and CoA must be available for reaction to proceed.

=**Thiamine**=


 * Also known as Vitamin B1
 * Makes up a thiamine pyrophosphate (TPP)
 * Structurally, it’s a pyrimidine attached to a methyl, a thiozole, and 2 phosphates
 * Thiozole contains the carbon that accepts the acetyl group
 * Essential for metabolizing glucose
 * Co-enzyme for the decarboxylation of α-keto acids such as pyruvic acid and α-keto glutaric acid
 * Also a cofactor for transketolase
 * Pyruvate is converted to Acetyl-CoA by oxidative decarboxylation
 * α-ketoglutarate is converted to succinyl-CoA by oxidative decarboxylation
 * Nutritional sources: pork, whole-grain cereals, legumes, and enriched grain productions
 * Storage is limited as liver stores can be depleted in 12-14 days

**Thiamine Deficiency**

 * Deficiency of Thiamine can lead to disturbances in carbohydrate metabolism
 * Decreased transketolase activity, particularly in erythrocytes and leukocytes
 * Lead to cardiovascular and neurologic lesions and emotional disturbances
 * Beri-Beri
 * Develop neuropathy, fatigue
 * “Dry” and “Wet” types; “Wet” Beri-Beri is more severe
 * Wernicke-Korsakoff Syndrome
 * Can result from alcoholism-related nutrient deficiency

=**Lipoic Acid**=


 * Also called Thioctic acid
 * Essential component in metabolism
 * Not a dietary requirement
 * Co-factor is hydrogen-transfer reactions
 * Key role in oxidative decarboxylation of pyruvate to Acetyl-CoA and α-ketoglutarate to succinyl-CoA
 * Carboxyl group usually bound to an enzyme by an amide bond

=**Co-enzyme A**=


 * Universal carrier of acyl groups
 * Forms high-energy, activated thioesters
 * High acetyl transfer potential because of hydrolysis of thioester is more thermodynamically favorable than oxygen ester.
 * ΔG°’ = -7.5 kcal/mol
 * Contains a Pantetheine attached to a 3’Pi-ADP
 * Pantetheine is made up by β-mercaptoethylamine and pantothenate
 * β-mercaptoethylamine contains the reactive –SH group
 * Pantothenate or pantothenic acid is one of the Vitamin B’s
 * Cannot be synthesized and must be acquired in diet
 * No known human disease due to deficiencies

=**Coenzyme NAD+**=


 * NAD+ can be reversibly reduced to the NADH+H+ form
 * NAD+ consist of a nicotinamide component bound to a ribose phosphate and an AMP component
 * The nicotinamide carbon #4 is the carbon accepting the hydride H- ion when it is reduced
 * Nicotinamide is an amine derivative of niacin or nicotinic acid
 * Deficiencies in niacin cause pellagra
 * Symptoms: dermatitis, diarrhea, and dementia
 * A related particle called NADP+ also contains nicotinamide and, like NAD+, is a co-enzyme of many oxidoreductases
 * NAD+ participates in many catabolic reactions
 * NADP+ participates in biosynthesis reactions

**Function of NAD+**

 * A Hydrogen acceptor in oxidation of HC=OH to C=O
 * Hydrogen from same carbon
 * Oxidation via oxidative phosphorylation yields 2.5 ATP

=**Coenzyme FAD**=


 * FAD or flavin adenine dinucleotide is a co-enzyme of most flavoproteins.
 * Tightly bound to enzyme
 * Contains Vitamin B2 or Riboflavin
 * No major human disease associated with riboflavin deficiency
 * Component of prosthetic groups FMN and FAD of flavoprotiens
 * Consists of an AMP component attached to a C5 ribitol and a isolloxazine ring components
 * Reduction site is the #1 and #5 nitrogen of the isoalloxazine
 * FMN or Flavin mononucleotide is a 5’-phosphate derivative of riboflavin
 * FAD = FMN+AMP

**Function of FAD**

 * FAD is generally a hydrogen acceptor in forming C=C bonds
 * Hydrogens from adjacent carbons
 * Oxidation via oxidative phosphorylation yields 1.5 ATP

=**Anaplerotic Reactions**=


 * Intermediates such as oxaloacetate are required to keep the TCA cycle going.
 * Pyruvate + CO2 + ATP --> Oxaloacetate + ADP + Pi
 * Catalyzed by pyruvate carboxylase with biotin as a co-enzyme
 * Activated by Acetyl-CoA
 * Occurs when TCA cycle is deficient in oxaloacetate

=**Amphibolic Nature of TCA Cycle**=
 * Glutamate + Pyruvate <--> α-ketoglutarate + alanine
 * Catalyzed by transaminases
 * Aspartate + Pyruvate <--> oxaloacetate + alanine
 * Catalyzed by transaminases
 * Citrate can be converted to Acetyl-CoA
 * Citrate can be transported out of mitochondria where it is converted to Acetyl-CoA and Oxaloacetate by ATP-citrate lysase
 * Acetyl-CoA can be further converted to fatty acids
 * Succinyl-CoA can be converted to heme synthesis

=**Metabolism of Amino Acids**=
 * Methionine, serine, cysteine, threonine, and glycine can be converted to pyruvate
 * Tryptophan can be converted to alanine which can be converted to pyruvate
 * Transaminease can remove the –NH2 from alanine to convert o pyruvate
 * Tyrosine and phenylalanine can be converted to fumarate
 * Isoleucine. Methionine, and valine can be converted to succinyl-CoA
 * Histidine, proline, hydroxyproline, and arginine can be converted to glutamic acid
 * Glutamic acid can be converted to α-ketoglutarate

**Other Reactions**

 * Oxaloacetate can be changed to P-Enol-Pyruvate by P-Enol Pyruvate carboxykinase
 * P-Enol-Pyruvate can be converted back to glucose
 * Propionate can be converted to succinyl CoA
 * Pyruvate can be converted to acetyl CoA

=**Regulation of TCA Cycle**=
 * Rate of Acetyl CoA formation from pyruvate is regulated by increasing Ca2+, ATP, NADH+H+, and Acetyl CoA
 * Isocitrate dehydrogenase catalyzes the committed step in Reaction 3
 * Reaction requires ADP as a positive or stimulatory allosteric modulator
 * Reactions 3 and 4 are inhibited allosterically by ATP and NADH+H+ and stimulated by increased Ca2+
 * In general, the TCA cycle is regulated by the need for ATP, the presence of O2, NAD+, FAD, and presence of TCA cycle intermediates such as oxaloacetate