Carbohydrate Metabolism II

The Carbohydrate Metabolism II section includes the citric acid cycle and electron transport chain which provides High Yield information for the MCAT, Medical School, Residency, and in the future career as a Physician. Prepare and Learn Ahead! Educating, Preparing, and Proving high-yield content, quizzes, and medical resources. to students who are interested in the medical field.

Acetyl-CoA

• Citric Acid Cycle/Krebs Cycle/tricarboxylic acid (TCA) cycle: occurs in mitochondria and functions to oxidize Acetyl-CoA to CO₂ and H₂O
  • Cycle produces high-energy molecules: NADH & FADH₂
• Acetyl-CoA can be obtained from the metabolism of carbohydrates, fatty acids and amino acids

Methods of Forming Acetyl-CoA

• Pyruvate dehydrogenase complex: a multienzyme compound that catalyzes the reactions which involved pyruvate entering the mitochondrion and subsequently be oxidized and decarboxylated
• Three carbon pyruvate is cleaved into a two-carbon acetyl group and a carbon dioxide
• Irreversible reaction (glucose cannot be formed directly from Acetyl-CoA
• In mammals, the complex is made up of five enzymes:
  • Work together to convert pyruvate to Acetyl-CoA
    • Pyruvate dehydrogenase (PDH)
    • Dihydrolipoyl transacetylase
    • Dihydrolipoyl dehydrogenase
  • Regulate the actions of PDH
    • Pyruvate dehydrogenase kinase
    • Pyruvate dehydrogenase phosphatase
• The overall reaction is exergonic: negative delta G, and is inhibited by an accumulation of Acetyl-CoA and NADH.
• Coenzyme A (CoA): written as CoA-SH to show that it is a thiol (with an –SH group)
  • Acetyl-CoA forms from a covalent attachment between the acetyl group and the –SH group. Results in the formation of a thioester
  • Thioesters are high-energy compounds that are necessary to drive other reactions forward.
• Pyruvate Dehydrogenase (PDH): pyruvate is oxidized to yield CO₂, and the remaining two-carbon molecules bind covalently to thiamine pyrophosphate (Vitamin B₁ or TPP)
  • TPP is a coenzyme that is non-covalently bonded to TPP
  • Mg²⁺ is also required in this reaction
• Dihydrolipoyl Transacetylase: a two-carbon molecule that is bound to TPP is oxidized and transferred to lipoic acid
  • Lipoic acid is a coenzyme that is covalently bonded to the enzyme
    • Disulfide group acts as an oxidizing agent, which creates the acetyl group
  • Acetyl group is bonded to lipoic acid by a thioester linkage
  • Dihydrolipoyl transacetylase then catalyzed the reaction involving CoA-SH & the newly formed thioester link
    • Causes the transfer of an acetyl group, so that it forms Acetyl-CoA
  • Lipoic acid is left in reduced from
• Dihydrolipoyl dehydrogenase: Uses the coenzyme Flavin adenine dinucleotide (FAD) to reoxidize the lipoic acid.
  • Lipoic acid oxidation results in FAD being reduced to FADH₂
    • FADH₂ is reoxidized to FAD by reducing NAD⁺ to NADH
• Glycolysis is the main contributor to the production of Acetyl-CoA, however, other pathways are also present.
  • The end goal is always the same: produce Acetyl-CoA so that it can be fed into the citric acid cycle
• Fatty Acid Oxidation (b-Oxidation): Activation causes a thioester bond to form between the carboxyl group of fatty acids and CoA-SH
  • Activated fatty acyl-CoA cannot cross the inner mitochondrial membrane
    • This is circumvented by having the fatty acyl group transferred to carnitine via a transesterification reaction
  • Once acyl-carnitine crosses the membrane, the fatty acyl group can be transferred to a mitochondrial CoA-SH
    • Carnitine’s main function is to carry acyl group from cytosolic CoA-SH to a mitochondrial CoA-SH
  • Beta-oxidation can occur once Acetyl-CoA is formed in the mitochondrial matrix
    • Removes two-carbon fragments from the carboxyl end.
• Amino Acid catabolism: Amino acid loses its amino group via transamination
  • Carbon skeletons can then form ketone bodies
  • Only certain amino acids can do this, these are termed: ketogenic
• Ketones: Acetyl-CoA is usually used to produce ketones when the pyruvate dehydrogenase complex is inhibited. Reverse reaction can occur as well
• Alcohol: Enzymes alcohol dehydrogenase & acetaldehyde dehydrogenase can convert small amounts of alcohol to Acetyl-CoA
  • Accompanied by NADH buildup, which inhibits the Krebs cycle
    • Thus Acetyl-CoA formed using this method is usually used to synthesize fatty acids.

Reactions of the Citric Acid Cycle

• Citric acid cycle takes place in the mitochondrial matrix
• Begins with the coupling of Acetyl-CoA to a molecule of oxaloacetate
  • Some parts of this molecule are oxidized to carbon dioxide, energy (GTP), and energy carriers (NADH and FADH₂)
  • Other substrates and products of the cycle are reused over and over again
• Oxygen is not directly required, but process will not occur anaerobically since a lack of oxygen would cause NADH and FADH₂ to build up and inhibit the cycle.

Key Reactions

Step 1 – Citrate Formation

• Acetyl-CoA and oxaloacetate undergo a condensation reaction to form citryl-CoA
• Hydrolysis of citryl-CoA yields citrate and CoA-SH
  • Catalyzed by citrate synthase
    • Synthases form new covalent bonds without needing significant energy
  • This step energetically favors the formation of citrate and helps move the cycle forward.
• 

Step 2 – Citrate Isomerized to Isocitrate

• Achiral citrate is isomerized to one of four possible isomers of isocitrate
  • Citrate binds at three points to the enzyme aconitase
  • Water is then lost from citrate to yield cis-aconitate
  • Water is then added back to form isocitrate
• Results in the switching of hydrogen and a hydroxyl group which is necessary to facilitate the subsequent oxidative decarboxylation
• An enzyme used is a metalloprotein which requires Fe²⁺
• 

Step 3 – a-Ketoglutarate and CO₂ Formation

• Isocitrate is oxidized to oxalosuccinate by isocitrate dehydrogenase
• Oxalosuccinate is decarboxylated to produce a-Ketoglutarate and CO₂
• Isocitrate dehydrogenase is the rate-limiting enzyme of the citric acid cycle
• One carbon is lost in this and the first NADH is produced from the intermediates of the cycle.
• 

Step 4 – Succinyl-CoA and CO₂ Formation

• Reactions are carried out by the a-Ketoglutarate dehydrogenase complex
• Similar mechanism, cofactors, and coenzymes to PDH complex
• a-Ketoglutarate and CoA come together to produce a molecule of Carbon dioxide
• Carbon dioxide is the last carbon lost from the cycle
• NAD⁺ is reduced to NADH in this step

Step 5 – Succinate Formation

• Thioester bond on succinyl-CoA is hydrolyzed to form succinate and CoA-SH
  • Coupled to the phosphorylation of GDP to GTP
  • The reaction catalyzed by succinyl-CoA synthetase
    • Synthetases create new covalent bonds with energy input
• Hydrolysis of the thioester bond releases a large amount of energy, which powers the phosphorylation of GDP.
• Nucleoside diphosphate kinase catalyzes a phosphate transfer from GTP to ADP
  • This is the only ATP that is produced through the citric acid cycle, rest is produced in the electron transport chain

Step 6 – Fumarate Formation

• Only step that does not take place in the mitochondrial matrix, occurs on the inner membrane
• Succinate undergoes oxidation to yield fumarate
  • Reaction catalyzed by succinate dehydrogenase
• Succinate dehydrogenase is a flavoprotein since it is covalently bonded to FAD (electron acceptor)
  • Enzyme is an integral protein on the inner mitochondrial membrane
• FAD is reduced to FADH₂ during this reaction
  • Each molecule of FADH₂ pass the electrons that it carries to the electron transport chain. (Indirectly gives rise to the production of 1.5 ATP)
• FAD is electron acceptor since Succinate is not a powerful enough reducing agent to reduce NAD⁺

Step 7 – Malate Formation

• Hydrolysis of alkene bond in fumarate, which produces malate
  • Catalyzed by fumarase
• Only L-malate forms from this reaction, even though it is possible for D-malate to form

Step 8 – Oxaloacetate Formed Anew

• Malate is oxidized to oxaloacetate
  • Catalyzed by malate dehydrogenase
• Third molecule of NAD⁺ is reduced to NADH during this step
• Oxaloacetate can then be used in the cycle again, and the maximum amount of high energy electron carriers have been produced
• 

Regulation

Pyruvate Dehydrogenase Complex Regulation

• Citric acid cycle can be regulated upstream from its actual starting point. This happens when PDH is phosphorylated
  • Phosphorylation is catalyzed by pyruvate dehydrogenase kinase
• Phosphorylating PDH inhibits Acetyl-CoA production
• PDH complex can be reactivated by the enzyme pyruvate dehydrogenase phosphatase
  • Occurs in response to high levels of ADP
  • This removes a phosphate from PDH, which then allows the reactivation of acetyl—CoA production
• Acetyl-CoA also uses a negative feedback effect on its own production
  • A high-fat diet is rich in Acetyl-CoA, which means that it is not necessary to produce Acetyl-CoA through carbohydrate metabolism
• ATP and NADH also tell the cell that there is a satisfactory amount of energy, and this inhibits PDH

Control Points of the Citric Acid Cycle

• Citrate Synthase: ATP and NADH function as allosteric inhibitors of citrate synthase
  • Both are products of the enzyme
  • Citrate and succinyl-CoA also allosterically inhibits citrate synthase
• Isocitrate dehydrogenase: This enzyme is inhibited by energy products: ATP and NADH
  • ADP and NAD⁺ act as allosteric activators by enhancing its affinity for substrates
• a-Ketoglutarate dehydrogenase complex: Reaction products of succinyl-CoA and NADH inhibit this enzyme complex
  • ATP also inhibits and slows the rate of the cycle when the cell has high levels of ATP
  • Stimulated by ADP and calcium ions

The Electron Transport Chain

• Final pathway that utilizes the harvested electrons from the different fuels of the body
• Proton gradient that this chain produces is what ultimately produces ATP
• Aerobic is the most efficient way of getting energy since it is conducted in the mitochondria. Anaerobic (such as glycolysis and fermentation) is conducted in the cytosol
• Mitochondria’s is specifically designed to harvest energy:
  • Citric acid cycle takes place in the mitochondrial matrix
  • Assemblies needed for oxidative phosphorylation is housed in the inner membrane
  • Inner membrane is assembled into folds called cristae (maximize surface area)
    • Essential for generating ATP through the proton-motive force
• Final step in aerobic respiration is two steps:
  • Electron transport along the inner mitochondrial membrane
  • Generation of ATP via ADP phosphorylation
• Overall process of aerobic respiration:
  • Electron rich molecules, NADH & FADH₂, transfer electrons to carrier proteins in the inner mitochondrial membrane
  • Electrons are given to oxygen in the form of hydride ions (H^–) and H₂O is formed
  • Simultaneously, energy is released from transporting electrons facilitates proton transport at three different locations.
    • Protons moved from mitochondrial matrix into the intermembrane space of the mitochondria (creates a greater concentration of hydrogen ions, which is used to drive ATP production)

Electron Flow and Complexes

• Formation of ATP requires energy (endergonic). This energy is provided by the exergonic electron transport pathway. I.e. – Two reactions are coupled
• Molecule with higher potential will be reduced, and other molecule will be oxidized. This reduction potential is what drives the transfer of electrons
  • NADH is a good electron donor and oxygen is a good electron acceptor

Complex I (NADH-CoQ oxidoreductase)

• Involves the transfer of electrons from NADH to coenzyme Q
• Complex has over twenty subunits, but two important ones are:
  • Protein that has an iron-sulfur cluster
  • Flavoprotein that oxidize NADH
    • Has a coenzyme (Flavin mononucleotide) that is covalently bonded to it
    • FMN is similar in structure to FAD (Flavin adenine dinucleotide)
• First step involves NADH transferring its electrons over to FMN
  • NADH oxidized to NAD⁺ and FMN is reduced to FMNH₂
• Flavoprotein then becomes reoxidized by reducing the iron-sulfur subunit
• Reduced iron-sulfur subunit donates its electrons that it received from FMNH₂ to coenzyme Q
  • CoQ becomes CoQH₂
• Proton pumping occurs at this site: four protons are moved into the intermembrane space

Complex II (Succinate-CoQ oxidoreductase)

• Transfers electrons to coenzyme Q (ubiquinone)
  • Receives electrons from succinate instead of NADH (like in complex I)
• Succinate is oxidized by FAD (FAD is converted FADH₂)
  • Succinate dehydrogenase is also responsible for this (like in citric acid)
• FADH₂ then gets reoxidized when it reduces an iron-sulfur protein
• Iron-sulfur protein is then reoxidized by reducing CoQ
• No hydrogen pumping occurs here (Does not contribute to proton gradient)
• succinate + CoQ + 2H+ -> fumarate + CoQH2

Complex III (CoQH₂-cytochrome C oxidoreductase)

• Facilitates the transfer of electrons from CoQ to cytochrome c
• Two separate complexes in the drawing are meant to illustrate the two subsequent reactions that takes place. Actually occur in the same complex
• Involves the reduction and oxidation of cytochromes
  • Proteins with heme groups where iron Is reduced to Fe²⁺ and reoxidized to Fe³⁺
• Only one electron is transferred per reaction, so two cytochromes are needed per CoA
• Main contribution of complex 3 is through the Q Cycle:
• Two electrons are shuttled from a molecule of ubiquinol (COQH₂), near the intermembrane space, to a molecule of ubiquinone (CoQ) near the mitochondrial matrix.
  • Two different electrons are attached to heme moieties, which reduces two molecules of cytochrome c
    • Assisted by sulfur/iron carrier
  • Through the Q cycle, four protons are displaced into the intermembrane space
• Q cycle increases the gradient of the proton-motive force across the inner membrane

Complex IV (Cytochrome c oxidase)

• Facilitates the culminating step of the electron transport chain
  • The transfer of electrons from cytochrome c to oxygen
• Contains subunits of cytochrome a, cytochrome a₃, and Cu²⁺ ions
  • Two cytochrome subunits make cytochrome oxidase
• Cytochrome oxidase is oxidized as oxygen is reduced to form water
• Two protons are moved across the membrane
• 

The Proton-Motive Force

• As [H⁺] increases in the intermembrane space:
  • pH drops in the intermediate space
  • Voltage difference, between the intermembrane space and matrix, increases due to proton pumping from electron transport chain
• Above two changes contribute to an electrochemical gradient: a gradient that has both chemical and electrostatic properties
  • Referred to as the proton-motive force since it is driven by proton transfer
  • Gradient stores energy and is responsible for ATP synthase

NADH Shuttles

• Net ATP yield per glucose is between 30-32
  • This is a range since the efficiency of aerobic respiration varies between cells
  • Variable efficiency is caused by the fact that cytosolic NADH cannot freely cross into the mitochondrial matrix
• NADH must find alternative forms of transport: referred to as shuttle mechanisms
  • Transfers the high-energy electrons of NADH to a carrier that can cross the inner mitochondrial membrane
    • ATP output (either 1.5 or 2.5) depends on shuttle mechanism used

Glycerol 3-Phosphate Shuttle

• Cytosol contains one isoform of glycerol-3-phosphate dehydrogenase
  • This oxidizes cytosolic NADH to NAD⁺ and forms glycerol 3-phosphate from DHAP
• Outer face of inner mitochondrial membrane has another isoform of glycerol-3-phosphate dehydrogenase that is FAD-dependent
  • Mitochondrial FAD is the oxidizing agent and this is reduced FADH₂
• FADH₂ transfers its electrons to the ETC via complex II
  • Generates 1.5 ATP for every molecule of cytosolic NADH

Malate-aspartate shuttle

• Cytosolic malate dehydrogenase catalyzes the reduction of cytosolic oxaloacetate to malate
  • Coupled with this reaction is the oxidation of NADH to NAD⁺
• Malate is able to cross the membrane into the matrix and then reverse the above reaction by the enzyme malate dehydrogenase
  • NADH formed from this reverse reaction can pass along its electrons to the ETC through complex 1
  • Generates 2.5 ATP
• Recycling of malate:
  • Malate oxidizes to oxaloacetate by malate dehydrogenase
  • Oxaloacetate is transaminated to form aspartate (aspartate transaminase)

Oxidative Phosphorylation

• ATP synthase is a protein complex that spans the entire inner mitochondrial membrane and protrudes into the matrix.

Chemiosmotic Coupling

• F₀ Portion: portion of ATP synthase that spans the membrane
  • Proton-motive force interacts with this portion
  • Functions as an ion channel: protons travel through F₀, along their gradient, and back into the matrix.
• Chemiosmotic Coupling: a process which allows the chemical energy of a gradient to be harnessed. Describes a direct relationship between the proton gradient and ATP synthesis.
  • i.e. – The ETC generates a high concentration of protons in the intermembrane space, the protons then flow into the matrix through the F₀ ion channels, and this release of energy is harnessed for the phosphorylation of ADP to ATP
• F₁ Portion: utilizes the energy released from the gradient to phosphorylate ADP to ATP
• Conformation Coupling: an alternative pathway that says that the mechanism between proton gradient and ATP synthesis is indirect
  • States that ATP is released by the synthase as a result of conformation changes that are caused by the gradient
  • F₁ portion is like a turbine: spins to facilitate the harnessing of energy
  • Less scientifically accepted
• -220 kJ/mol of energy from the exergonic proton-motive force dissipation. Energy is used to drive phosphorylation reaction

Regulation

• O₂ and ADP are the key regulators of oxidative phosphorylation
• If O₂ is limited, then the rate of oxidative phosphorylation decreases and the concentration of NADH and FADH₂ increases
  • Accumulation of NADH inhibits the citric acid cycle
• Respiratory Regulation: coordinated regulation of the different pathways that are involved
• If there is adequate oxygen available, then the rate of oxidative phosphorylation is dependent on the availability of ADP
• ADP allosterically activates isocitrate dehydrogenase
  • This increases the rate of the citric acid cycle and the production of electron carrying compounds: NADH & FADH₂
  • Enzyme’s cause an increase in the rate of electron transport and ATP synthesis.