Metabolism and Energy Production

Metabolism and Energy Production

Metabolism

• Metabolism: The sum of all chemical reactions involved in maintaining the dynamic state of a cell or organism.
• Pathway: A series of biochemical reactions.
• Metabolism is the sum of catabolism and anabolism.
• Metabolic pathways

Catabolism

• Catabolism: The process of breaking down large nutrient molecules into smaller molecules with the concurrent production of energy.
• The purpose of the catabolic pathway is to convert the energy stored in food molecules into energy stored in molecules of ATP (energy carrier).

Anabolism

• Anabolism: The process of synthesizing larger molecules from smaller ones.
• Catabolism involves the breakdown of larger molecules (Polysaccharides, Triglycerides, Proteins) into smaller ones (Fatty acids and glycerol, Monosaccharides, Amino Acids) via oxidation and releases energy.
• Anabolism involves using energy and reducing agents to synthesize larger molecules (Proteins and nucleic acids) from smaller ones (Amino Acids, Some nutrients and products of catabolism).
• Excretion: Removal of waste products from both catabolism and anabolism.

Metabolic Pathways

• Metabolism is a series of consecutive reactions called a metabolic pathway, which can be linear or cyclic.

• Linear metabolic pathway: A → B → C → D

• Cyclic metabolic pathway:

   A → B
   ↑     ↓
   D ← C
  

Mitochondria

• Mitochondria: Organelles in which the common catabolic pathway takes place in higher organisms. The enzymes that catalyze catabolic pathways are all located in these organelles.
• IMS: intermembrane space

Catabolism - Stage 1: Digestion

• The catabolism of food begins with digestion, which is catalyzed by enzymes in the saliva, stomach, and small intestines.
• Carbohydrates are hydrolyzed into monosaccharides beginning with amylase enzymes in saliva and continuing in the small intestine.
• Proteins are hydrolyzed into amino acids:
  • Stomach: Pepsin
  • Intestines: Trypsin and Chymotrypsin, Elastase, Carboxypeptidase A, B, Aminopeptidase, Dipeptidase, Tripeptidase
  • Polypeptides are broken down into oligopeptides, then into amino acids which are absorbed.
• The protease pepsin begins to hydrolyze proteins to amino acids in the stomach.
• Reaction: $protein + H_2O \xrightarrow{pepsin} amino acid$
• Triacylglycerols are emulsified by bile secreted by the liver, then hydrolyzed by lipases in the small intestines into 3 fatty acids and a glycerol backbone.

Catabolism Overview - Stages 2, 3, and 4

• Stage 2: Formation of Acetyl-CoA
• Stage 3: TCA cycle (Citric Acid Cycle)
• Stage 4: Oxidative Phosphorylation
• Proteins are broken down into amino acids. Amino acids are converted into Acetyl-CoA. Ammonia is released as a byproduct.
• Polysaccharides are broken down into glucose. Glucose goes through glycolysis to produce pyruvate, which is then converted to Acetyl-CoA.
• Triacylglycerols are broken down into fatty acids and glycerol. Fatty acids undergo beta-oxidation to produce Acetyl-CoA.
• Acetyl-CoA enters the Citric Acid Cycle, producing $CO<em>2$, NADH, and $FADH</em>2$.
• NADH and $FADH_2$ then go through oxidative phosphorylation to produce ATP.

Location of Metabolic Processes

• TCA cycle and Oxidative phosphorylation take place in the mitochondria.

The Common Metabolic Pathway

• The common catabolic pathway has two parts:
  • The citric acid cycle, also called the tricarboxylic acid (TCA) or Krebs cycle.
  • Electron transport chain and ATP synthase (phosphorylation), together called oxidative phosphorylation pathway.
• Oxidative Phosphorylation: Electron Transport Chain + ATP Synthase

Principal Compounds in the Common Catabolic Pathway

• Four principal compounds participate in the common catabolic pathway. They are:
  • AMP, ADP, and ATP: all three are agents for the storage and transfer of phosphate groups.
  • $NAD^+$/NADH: they are coenzymes for the transfer of electrons in biological oxidation-reduction reactions.
  • FAD/$FADH_2$: they too are coenzymes for the transfer of electrons in biological oxidation-reduction reactions.
  • Coenzyme A; abbreviated CoA or CoA-SH: An agent for the transfer of acetyl groups.

Adenosine Triphosphate (ATP)

• ATP is the most important compound involved in the transfer of phosphate groups and energy.
• ATP contains two phosphoric anhydride bonds and one phosphoric ester bond.
• Hydrolysis of the terminal phosphate (anhydride) of ATP gives ADP, hydrogen phosphate ion, and $7.3 kcal/mol$ of energy.
• Phosphorylation is the reverse reaction, where a phosphate group is added to ADP, forming ATP requiring $7.3 kcal/mol$ of energy.

Coupled Reactions

• Coupling an energetically unfavorable reaction with a favorable one that releases more energy than the amount required is common in biological reactions.

$NAD^+$/$NADP^+$

• Nicotinamide Adenine Dinucleotide (Phosphate) is a biological oxidizing agent (a coenzyme).
• The + on $NAD^+$/$NADP^+$ represents the + charge on nitrogen.

Reduction of $NAD(P)^+$ to $NAD(P)H$

• NADH/NADPH is an electron and hydrogen ion ($H^+$) transporting molecule.
• Obligate 2 e- transfer.
• $NAD^+ + H:^- \rightarrow NADH$

FAD/$FADH_2$

• Flavin Adenine Dinucleotide (FAD) is also a biological oxidizing agent. The operative part if the Flavin portion.

FAD/$FADH_2$

• FAD is a two-electron oxidizing agent and is reduced to $FADH<em>2$. $FADH</em>2$ is a two-electron reducing agent and is oxidized to FAD.
• Only the flavin moiety is shown in the structures.

Coenzyme A (CoA or CoA-SH)

• Coenzyme A (CoA) is an acetyl-transporting molecule.
• The business end is the -SH (sulfhydryl) group at the left end.
• The acetyl group of acetyl CoA is bound as a high-energy thioester bond; releases $7.51 kcal/mol$ energy when hydrolyzed. B Vitamin.

Citric Acid Cycle - Carbon Balance

• Simplified view showing only the carbon balance.
• A 2-carbon molecule enters the cycle.
• Two molecules of $CO_2$ are released.
• Cycle returns to the starting 4-carbon molecule.

Citric Acid Cycle

• A more detailed view of the Krebs cycle showing that the fuel for the cycle is the two-carbon acetyl group of acetyl CoA and that with each turn of the cycle two carbons are released as $CO_2$.

Reactants and Products of Citric Acid Cycle

• Carbohydrates, Fats, and Proteins are broken down into Monosaccharides, Fatty acids, and Amino acids, respectively.
• These are converted to Acetyl coenzyme A ($CH_3–CO—S—CoA$).
• Acetyl coenzyme A reacts with Oxaloacetate ($H<em>2C-COO^-\ \ \ \ \ O=C-COO^-\ \ \ \ \ H</em>2C-COO^-$) to form Citrate ($H<em>2C-COO^-\ \ \ \ \ HO-C-COO^-\ \ \ \ \ H</em>2C-COO^-$).
• Citrate is converted to Isocitrate ($H_2C-COO^-\ \ \ \ \ H-C-COO^-\ \ \ \ \ HO-C-H\ \ \ \ \ COO^-$).
• Isocitrate is converted to α-Ketoglutarate ($H<em>2C-COO^-\ \ \ \ \ 0=C-COO^-\ \ \ \ \ H</em>2C^-\ \ \ \ \ COO^-$).
• α-Ketoglutarate is converted to Succinyl CoA ($H<em>2C-COO^-\ \ \ \ \ H</em>2C^-\ \ \ \ \ H_2C\ CO-S-COA$).
• Succinyl CoA is converted to Succinate ($H<em>2C-COO^-\ \ \ \ \ H</em>2C^-\ \ \ \ \ H_2C-COO^-$).
• Succinate is converted to Fumarate ($\ HC-COO^-\ \ \ \ \ OOC-CH$).
• Fumarate is converted to Malate ($HO-C-H\ \ \ \ \ CH<em>2\ \ \ \ \ CO</em>2^-$).
• Malate is converted back to Oxaloacetate.
• Products include: NADH + H+, FADH2, GTP, and CO2

Citric Acid Cycle Steps

• Step 1: Acetyl CoA enters the cycle by combining with a C4 compound, oxaloacetate. This step is catalyzed by the enzyme citrate synthase.
• Step 2: Isomerizes the $3^o$ alcohol in citrate to the $2^o$ alcohol in isocitrate; it is catalyzed by aconitase.
• The citrate ion is dehydrated to cis-aconitate which is then hydrated to Isocitrate instead of citrate.
• Citrate and aconitate are achiral.
• Isocitrate is chiral; it has stereocenters and thus stereoisomers are possible. But only one of the possible stereoisomers is formed in the cycle due to the spcificity of aconitase.
• Step 3: The isocitrate formed is oxidized and then decarboxylated at the same time to produce a C5 compound known as α-ketoglutarate. Enzyme: Isocitrate dehydrogenase
• Steps 4: Releases another $CO_2$ with the oxidation of α- ketoglutarate by $NAD^+$ in the presence of coenzyme A to form succinyl CoA and NADH. α-ketoglutarate dehydrogenase
• Steps 5: The thioester bond of succinyl CoA is hydrolyzed to form succinate, releasing energy that converts GDP to GTP. The chemical energy stored in GTP (in the form of high-energy phosphoric anhydride bonds) is released during hydrolysis of GTP and drives many important biochemical reactions.
• Step 6: Succinate is oxidized by FAD to trans-fumarate.
• Step 7: The fumarate is now hydrated to give L-malate ion.
• Step 8: In the final step of the cycle, malate is oxidized to oxaloacetate which will react with acetyl CoA to start another round of the cycle by repeating Step 1.

Overall Reaction of the Citric Acid Cycle

• $CH<em>3COSCoA + 2 H</em>2O + GDP + 3 NAD^+ + HPO<em>4^{2-} + FAD \rightarrow 2 CO</em>2 + HSCOA + GTP + 3 NADH + 3 H^+ + FADH_2$
• Acetyl CoA + 2 water + GDP + 3 $NAD^+$ + $HPO<em>4^{2-}$ + FAD yields 2 carbon dioxide + HSCoA + GTP + 3NADH + 3$H^+$ + $FADH</em>2$

Citric Acid Cycle - Key Points

• Net effect: one two-carbon acetyl group enters the cycle (changes C4 to C6 in step 1) and two $CO_2$ are given off.
• How does the cycle produce energy? A high-energy molecule, GTP is produced. Energy is also produced in other steps that convert $NAD^+$ to NADH and FAD to $FADH_2$. They carry the $H^+$ and electrons that will provide energy for the synthesis of ATP.
• How is the cycle regulated? By a few feedback mechanisms. When the essential products of the cycle accumulate, they inhibit some of the enzymes in the cycle.
  • Inhibition: Citrate synthase (step 1), isocitrate dehydrogenase (step 3), α-ketogutarte dehydrogenase (step 4)

TCA Cycle in Catabolism

• The catabolism of proteins, carbohydrates, and fatty acids all feed into the citric acid cycle at one or more points.
• The end products are the reduced coenzymes NADH (3 per $C<em>2$ fragment) and $FADH</em>2$ (1 per $C_2$ fragment).

Oxidative Phosphorylation

• Electron Transport Chain + ATP Synthase
• Take place in mitochondria.
• Electrons move through Complexes I, II, III, and IV.
• Protons ($H^+$) are pumped from the matrix to the intermembrane space.
• $FADH_2$ is oxidized to FAD.
• NADH is oxidized to $NAD^+$.
• ATP Synthase (Complex V) uses the proton gradient to produce ATP from ADP.

Substrates for Oxidative Phosphorylation

• TCA cycle: $2 H^+ + \frac{1}{2} O<em>2 \rightarrow H</em>2O$
• The purpose of the electron transport or respiratory chain is:
  • (1) to pass along 2$H^+$ ions and 2e-, derived from NADH and $FADH_2$ produced in the TCA cycle, to combine with oxygen from respiration at the end of the chain;
  • (2) to drive the synthesis of ATP from ADP.

Electron and $H^+$ Transport

• The overall electron chain transport reaction is: $2 H^+ + 2 e^- + \frac{1}{2} O<em>2 \rightarrow H</em>2O + energy$
• The energy released is used to drive the synthesis of ATP molecule.

Oxidative Phosphorylation Pathway

• Schematic diagram of the electron and $H^+$ transport chain (subsequent phosphorylation is not shown here).
• The four enzyme complexes are all embedded in the inner membrane of the mitochondria.
• The enzymes are arranged in order of increased affinity for electrons, so electrons easily flow through the four enzyme complexes.

NADH electron-path

• FMN: flavin mononucleotide
• Some of the energy released in the oxidation of NADH to $NAD^+$ in Complex I is used to move the 4$H^+$ from the matrix into the intermembrane space.
• The energy released from oxidation of $FADH_2$ in Complex II is not sufficient to pump any protons across the membrane.
• Complex II oxidizes $FADH_2$ produced in the TCA cycle to FAD. The electrons produced from this oxidation are used to reduce Coenzyme Q.
• Coenzyme Q (CoQ) is soluble in lipids and can move laterally within the membrane.
• Some of the energy released in the redox reactions in Complex III is also used to pump 4 more $H^+$ from the redox reactions from the matrix into the intermembrane space.
• CoQ10
• Cytochrome c is a membrane protein that is mobile and can freely move in inner membrane space.
• Complex IV = cytochrome oxidase.
• During the redox reaction occurring in Complex IV, Some of the energy released is used to pump two more $H^+$ out of the matrix into the intermembrane space as well.

NADH/$FADH_2$ electron-path

• Overall:
  • 10 $H^+$ are pumped out per NADH (from Complexes I, III and IV)
  • Six $H^+$ are pumped out per $FADH_2$ (from Complexes III and IV)
• Why does $FADH_2$ only pump 6?

Chemiosmotic Pump

• Electron transport processes through complexes I, III, and IV are coupled to the transport of protons from the matrix to the intermembrane space.
• This establishes a proton gradient (or pH gradient) across the inner membrane.

Chemiosmotic Theory

• To generate ATP, however, these protons must be pumped back into the matrix to combine with $O_2$.
• Mitchell proposed that “the accumulation of protons ($H^+$) in the intermembrane space in turn creates an osmotic pressure, thus the term chemiosmotic theory”.
• According to Mitchell, the proton gradient creates a driving force (acting like a pump) that in turn drives the protons back to the mitochondrion to be used to generate ATP.
• This theory was confirmed by subsequent studies and Mitchell won the Nobel Prize in chemistry in 1978.
• Can $H^+$ cross the phospholipid bilayer freely?
• According to Mitchell, this transport is achieved because of the action of the fifth complex that is located on the inner membrane of the mitochondrion, the ATP synthase (proton translocating ATPase).
• Every 4 $H^+$ passing through complex 5 produces 1 ATP.

Properties of $F<em>1F</em>0$-ATP Synthase

• Proton-translocating ATP Synthase
  • Converts chemical energy (pH gradient) into mechanical energy
  • $F_1$: water-soluble peripheral membrane protein
    • Synthesizes ATP
  • $F_0$: water-insoluble transmembrane protein
    • Translocates protons
  • Transmembrane ring Rotation Driven by proton translocation

Chemiosmotic Theory - Summary

• The protons that enter mitochondrion combine with the electrons transported through the electron transport chain and with oxygen to form water.
• The energy released from the reaction is used to covert ADP to ATP in the $F_1$ unit of ATPase via the reaction:
• Overall Effect: one ATP is produced for every 4$H^+$ ions produced by the oxidative phosphorylation pathway.

Oxygen Function

• The many redox reaction taking place in the TCA cycle require oxygen.
• The oxygen we inhale, therefore, has two distinct functions:
  • It oxidizes NADH to $NAD^+$ and $FADH_2$ to FAD so that these molecules can return to participate in the citric acid cycle.
  • The production of water from oxygen provides the energy needed for the conversion of ADP to ATP.

Energy Yield

• The energy released during electron transport is now captured in the form of chemical energy of the ATP molecules.
• For each two-carbon acetyl unit ($C<em>2$ fragment) entering the citric acid cycle, we get three NADH and one $FADH</em>2$.
• For each NADH oxidized to $NAD^+$, we get 2.5 ATP.
• For each $FADH_2$ oxidized to FAD, we get 1.5 ATP.
• Thus, the energy balance for the entire common catabolic pathway (TCA + Ox. Phos.) produced per $C<em>2$ fragment oxidized to $CO</em>2$ is:
  • 3 NADH X 2.5 ATP/NADH = 7.5 ATP
  • 1 $FADH<em>2$ X 1.5 ATP/$FADH</em>2$ = 1.5 ATP
  • 1 GTP = 1 ATP
  • Total = 10 ATP

Energy Yield (Net Reaction)

• The net reaction involving a $C_2$ fragment can then be written as:
• $C<em>2 + 2O</em>2 + 10 ADP + 10 P<em>i \longrightarrow 10 ATP + 2 CO</em>2$
• The 10 ATP molecules now release their energy when they are converted to ADP in various metabolic reactions requiring energy.

Metabolism - Summary Diagram

• Diagram showing:
  • Citric Acid Cycle.
  • Electron Transport.
  • ATP production by ATP synthase.