1.1 Metabolism

Overview:

• Metabolism: the sum of all biochemical reactions within cells, managing the conversion of nutrients into energy and the synthesis of essential molecules.

• Biomolecules play a crucial role in metabolism, serving as 

  • substrates 

  • Catalysts

  • regulators of cellular processes.

• Metabolism is essential for sustaining life, as it provides the energy needed for cellular activities and facilitates the growth and maintenance of organisms.

Types of metabolism:

• Anabolism:

  • Building complex molecules from simpler ones.

  • Requires energy input.

  • Examples: protein synthesis and the formation of cell structures.

• Catabolism:

  • Breaking down complex molecules into simpler ones.

  • Releases energy.

  • Examples: cellular respiration and the digestion of food.

Metabolic Pathways:

• Series of interconnected chemical reactions that occur within a cell to convert substrates into products, often involving multiple enzymes.

• Metabolic pathways are interconnected, allowing the flow of molecules between them.

•  Metabolites serve as intermediates shared among different pathways.

• Catabolic Pathways:

  • Break down complex molecules into simpler ones.

  • Release energy, often in the form of ATP.

  • Example: Cellular respiration.

• Anabolic Pathways:

  •  Build complex molecules from simpler ones.

  •  Consume energy, often in the form of ATP.

  • Example: Protein synthesis.

• Central Metabolic Pathways:

  • Core pathways are shared by many organisms.

  •  Include glycolysis, the Krebs cycle, and oxidative phosphorylation.

Activation Energy:

• Activation energy: the energy required to initiate a chemical reaction and start the conversion of reactants into products.

• Represents the energy hurdle that molecules must overcome for a reaction to occur.

Factors affecting activation energy:

• Catalysts: They lower the activation energy by providing an alternative reaction pathway.

• Temperature: Increasing temperature generally increases the kinetic energy of molecules, making it easier to reach activation energy.

• Presence of Inhibitors: Inhibitors may increase the activation energy by interfering with the reaction pathway, slowing down the reaction.

• Concentration of Reactants: Higher concentrations provide more reactant molecules, increasing the likelihood of successful collisions and reducing activation energy.

Types of enzymatic reaction:

• Exergonic reaction:

  • Release energy to the surroundings.

  • The final state has lower energy than the initial state.

  • Common in catabolic processes like cellular respiration.

• Endergonic reaction:

  • Absorb energy from the surroundings.

  • The final state has higher energy than the initial state.

  • Typical in anabolic processes like protein synthesis.

1.2 Enzymes

Enzymes:

• Enzymes: enhance the rate of biological chemical reactions by lowering activation energy.

• They are biological catalysts.

• All enzymes are proteinaceous except ribozyme and ribonuclease.

• Enzymes show tertiary and quarternary structures and are very specific for biological activity.

• Most enzymes are found in mitochondria.

• Small enzyme: Peroxidase.

• Largest enzyme: Catalase

Nomenclature and Classification of Enzymes

• Nomenclature: suffix = ase

• Source of extraction: from where it is extracted.

• 6 classes of enzymes:

  • OTHLiL - anagram to remember

  • Oxidoreductase:

    • Enzymes that are involved in an oxidation-reduction reaction.

    • e.g.: Alcohol dehydrogenase, cytochrome oxidase.

  • Transferase:

    • Enzymes that catalyze reactions involved in the transfer of functional groups.

    • e.g.: hexokinase, trans-aminase.

  • Hydrolase:

    • Enzyme catalyzing hydrolysis of ester, ether, peptides, etc.

    • These enzymes break large molecules into smaller molecules by the introduction/presence of H₂O molecules.

  • Lyases:

    • They break specific covalent bonds and remove a group without hydrolysis, oxidation, etc.

    • e.g. Aldolase, fumarase.

  • Isomerase:

    • Rearrangement of molecular structure to form isomers.

  • Ligases:

    • The enzyme catalyzes the synthetic reaction where two molecules are joined together.

Types of Enzymes:

• Simple enzyme: consists of only proteins and catalyzes their substrate-specific reactions.

• Conjugate enzyme/Holo enzyme: Made up of protein and non-protein parts.

  • Protein part: Apoenzyme

  • Non-protein part: Co-factor

    • Organic:

      • Coenzyme: A coenzyme is a loosely bound/organic co-factor. It can be easily removed.

      • Prosthetic group: A prosthetic group is a tightly bound organic co-factor.

    • Inorganic: They form coordination bonds with the side-chain at the active site and the same time for one/more coordination bonds with substrate.

Mode of enzyme action

The hypothesis regarding the mode of enzyme action

• Lock and Key Hypothesis:

  • According to this theory:

    • Enzymes are rigid and pre-shaped.

    • Substrate fit to the active site just as a key fit into a proper lock.

• Induced fit hypothesis/ theory:

  • Proposed by Koshland.

  • The most accepted hypothesis based on enzyme action.

  • Enzymes are not rigid and pre-shaped.

Mechanism of enzyme action:

• Substrate → Product

• Lowering down of activation energy.

• Do not alter the equilibrium.

• Enzymes are biocatalyst.

Factors affecting enzyme action:

• Temperature:

  • at high temperature: denaturation

  • at low temperature: inactivation

  • optimum temperature: 25-40 degrees Celsius for enzymatic activity.

• pH:

  • optimum pH = enzyme activity very high.

  • enzymes:

    • endoenzyme (inside the cell)

    • exoenzyme (enzymes are synthesized inside the cell but secreted from the cell to work externally).

• Substrate concentration:

  • The enzyme is larger and bears several active sites with the increase in substrate concentration the velocity of the reaction rises first and the reaction reaches a maximum velocity. (V_max)

  • The velocity is not exceeded by any further rise in the concentration of substrate.

  • Michalis Menten Constant (K_m):

    • It is a mathematical derivation/constant that indicates the concentration of substrate at which reaction velocity reaches half of V_max.

    • K_m indicates the affinity of the enzyme for its substrate.

    • A high K_m indicates low affinity of enzyme and a low Km indicates high affinity.

    • Km is inversely proportional to turn over number.

    • Allosteric enzymes do not obey K_m.

Inhibitors:

•  Inhibitors: chemical molecules that inhibit enzyme activity.

• Inhibitors are of two types:

• Competitive inhibitors:

  • Inhibitors have a similar structure to the substrate.

  • They favor the lock and key hypothesis.

  • Reversible in nature.

  • K_m increases but V_max remains constant.

• Non-competitive inhibitors:

  • Some inhibitors do not compete for the active site of the enzyme but destroy the structure of the enzyme, the physical structure of the enzyme is altered as a result and does not form an enzyme-substrate complex.

  • They favor the induced-fit theory.

  • Irreversible in nature.

  • K_m remains constant but V_max changes.

1.3 Cell Respiration 

Energy Conversion: Mitochondria and Chloroplast

• Mitochondria: Occurs in all cells.

  • burn food particles to produce ATP by oxidative phosphorylation.

• Chloroplast: it occurs only in plants and green algae.

  • harness solar energy to produce ATP by photosynthesis.

The Mitochondria

• Mitochondria have an outer and inner membrane.

• Cristae: The inner mitochondrial membrane is folded to form invaginations called cristae.

• Intermembrane space: The narrow gap between the two membranes.

• The outer mitochondrial membrane is freely permeable to ions and small molecules.

• The inner mitochondrial membrane:

  •  diffusion barrier to ions and small molecules.

  • It also contains the machinery for electron transport and ATP synthesis.

• The membranes of the cristae:

  • They are continuous with the boundary membrane.

  • It contains the ATP synthase enzyme that produces most of the cell’s ATP.

  • They also contain the large protein complexes of the respiratory chain—the name of the mitochondrion’s electron-transport chain.

• The mitochondrial matrix contains enzymes that-

  • convert pyruvate and fatty acids to acetyl CoA 

  • oxidize this acetyl CoA to CO₂ through the citric acid cycle.

How do cells obtain energy from food?

• Glycolysis: The major process of oxidizing sugars is the sequence of reactions known as glycolysis.

  • It is common in both aerobic (with the presence of oxygen) and anaerobic (without the presence of oxygen) reactions.

  • It takes place in the cytoplasm of the cell.

  • It starts with 6-carbon glucose to finally result in two molecules of 3-C pyruvate. In plants, this glucose is derived from sucrose.

  • Total ATP produced: 8 ATP

• Fermentation:

  • Alcoholic Fermentation:

    • It occurs in yeast.

    • The process is hazardous as either acid or alcohol is produced. Yeasts poison themselves to death when the concentration reaches about 13%.

    • It yields ethyl alcohol as the final product.

    • 2 ATP molecules formed

• Lactic Acid Fermentation:

  • It occurs in the muscles of humans during an intense workout.

  • it yields lactic acid as the final product.

  • Total ATP produced: 2ATP

TCA (tricarboxylic acid cycle) or Kreb’s cycle or citric acid cycle

• The TCA cycle occurs in the mitochondrial matrix.

• All the enzymes of the TCA cycle, except succinate dehydrogenase (in the inner mitochondrial membrane) present in the matrix.

• During the Krebs cycle acetyl Co-A is completely oxidized into CO₂.

• Kreb’s cycle is also called the citric acid cycle because the first compound is citric acid.

1. In Kreb’s cycle oxaloacetic acid (OAA) is the first member and it also acts as the first acceptor of Acetyl Co-A.

2. The TCA cycle starts with the condensation of the acetyl group with oxaloacetic acid and water to yield citric acid. The reaction is catalyzed by the enzyme citrate synthase and a molecule of CoA is released.

3. Citrate is then isomerized to isocitrate. It is followed by two successive decarboxylations.

4. In the remaining steps of the citric acid cycle, succinyl Co-A is oxidized to OAA allowing the cycle to continue. During the conversion of succinyl Co-A to succinic acid, a molecule of GTP is synthesized.

5. Also, there are three points in the cycle where NAD+ is reduced to NADH + H⁺ and one point where FAD⁺ is reduced to FADH₂.

6. The continued oxidation of acetyl CoA via the TCA cycle requires the continued replenishment of oxaloacetic acid. In addition, it also requires the regeneration of NAD⁺ and FAD⁺ from NADH and FADH₂ respectively.

Oxidation or occurs at 4 places in one Krebs cycle, resulting in the formation of 3 NADH, and 1 FADH₂,  Along with 1 GTP produced by substrate-level phosphorylation in each turn of the TCA cycle equals 12 ATP.

Electron Transport System (ETS)/ Oxidative Phosphorylation OR Respiratory Chain & (Terminal Oxidation of NADH + H⁺ & FADH₂)

• All the reduced hydrogen acceptors like NADH + H⁺, and FADH₂ move to the Electron Transport System where they release their hydrogen and get reoxidized to NAD⁺ and FAD⁺ so that they can again enter into the respiration process.

• ETS: the chain of some hydrogen and electron carriers present in the inner mitochondrial membrane.

• The significance of ETS: to remove hydrogens from reduced hydrogen acceptors NADH + H⁺ & FADH₂. During this process, hydrogen acceptors get reoxidized and ATP is produced.

• Components of ETS are categorized as:

  • Complex I: (NADH dehydrogenase complex): FMN, Fe-S

  • Complex II: (Succinic dehydrogenase complex): FAD, Fe-S

  • Complex III: (Cytochrome bc1 complex): Cytochrome b-Cyt c1, Fe-S

  • Complex IV: (Cytochrome c oxidase complex): Cyt a and Cyt a3, 2 Cu centers.

  • Complex V: (ATP synthase/ ATPase/Oxysome): Fo (integral) - F1 (peripheral)

• Electrons from NADH and H⁺ produced in the mitochondrial matrix during the citric acid cycle oxidized by NADH dehydrogenase (complex I). Electrons are then transferred to ubiquinone located within the inner membrane.

• Ubiquinone also receives reducing equivalents via FADH₂ (complex II) generated during succinate oxidation in the citric acid cycle.

• The reduced ubiquinone (ubiquinol) is then oxidized with the transfer of electrons to cytochrome C via cytochrome BC1 complex (complex III).

• Cytochrome C: small protein attached to the outer surface of the inner membrane. Acts as a mobile carrier for the transfer of electrons between complex III and IV.

• Complex IV: the cytochrome C oxidase complex containing cytochromes a and a3, and two copper centers.

• When the electrons pass from one carrier to another via complexes I and IV in the electron transport chain, they are coupled to ATP synthase (complex V) for the production of ATP from ADP and inorganic phosphate.

• The number of ATP molecules synthesized depends on the nature of the electron donor. Oxidation of one molecule of NADH + H⁺ gives rise to 3 molecules of ATP while oxidizing one molecule of FADH₂ produces 2 molecules of ATP.

• Passage of 4H⁺ through F₀ particle or proton channel leads to synthesis of 1 ATP.

• Although aerobic respiration takes place only in the presence of oxygen, the role of oxygen is limited to the terminal stage of the process.

  • Yet, the presence of oxygen is vital, since it drives the whole process by removing hydrogen from the system. Oxygen acts as the final hydrogen acceptor.

• Unlike photophosphorylation, where light energy is utilized to produce the proton gradient required for phosphorylation, in respiration it is the energy of oxidation-reduction utilized for the same process. 

1.4 Photosynthesis

 Photosynthesis

• The process by which green plants, algae, and some bacteria convert light energy into chemical energy in the form of glucose.

• It primarily occurs in the chloroplast of plant cells.

• Reactants:

  •  Carbon dioxide (CO₂) and water (H₂O) are used as raw materials.

• Products:

  •  Glucose (C₆H₁₂O₆) and oxygen (O₂) are produced as a result of the photosynthetic process.

• Light-Dependent Reactions:

  • Occur in the thylakoid membrane.

  • Capture light energy and convert it into chemical energy (ATP and NADPH).

  • Generate oxygen as a byproduct.

• Light-Independent Reactions (Calvin Cycle):

  • Take place in the stroma of the chloroplast.

  • Utilize ATP and NADPH to convert carbon dioxide into glucose through a series of chemical reactions.

Chemiosmotic Process

• Stage 1:

  • High-energy electrons (derived from the oxidation of food molecules, from pigments excited by sunlight, or from other sources described later) are transferred along a series of electron-transport protein complexes that form an electron-transport chain embedded in a membrane.

  • Each electron transfer releases a small amount of energy used to pump protons and generate a large electrochemical gradient across the membrane.

• Stage 2:

  • The protons flow back down their electrochemical gradient through ATP synthase, catalyzing the production of ATP from ADP and inorganic phosphate (Pi).

• In this way, the energy derived from food or sunlight in stage 1 is converted into the chemical energy of a phosphate bond in ATP.

Chloroplast

• Photosynthesis occurs in a specialized intracellular organelle.

• They have

  • a highly permeable outer membrane

  • a much less permeable inner membrane, in which membrane transport proteins are embedded

  • a narrow intermembrane space in between.

• The inner chloroplast membrane surrounds a large space called the stroma, analogous to the mitochondrial matrix.

• The chloroplast has its genome and genetic system.

• The stroma therefore also contains a special set of ribosomes, RNAs, and chloroplast DNA.

• The electron-transport chains, photosynthetic light-capturing systems, and ATP synthase are all contained in the thylakoid membrane.

  • It is a separate distinct membrane that forms a set of flattened, disc-like sacs called thylakoids.

Light Reaction

• Light reactions or the photochemical phase include light absorption, water splitting, oxygen release, and formation of high-energy chemical intermediates like ATP and NADPH.

• Photosystems are required for this process.

• The groups of photosynthetic pigments in the thylakoid membrane are known as photosystems.

• The pigments are organized as two discrete photochemical light-harvesting complexes (LHC) within the photosystem I and photosystem II.

• In every photosystem there is a reaction centre surrounded by accessory pigments.

• The reaction center is different in both the photosystems.

  • In PS I the reaction center chlorophyll-a has an absorption peak at 700 nm, called P700.

  • In PSII it has an absorption peak at 680 nm, called P680.

• These are named in the sequence of their discovery, and not in the sequence in which they function during the light reaction.

The electron transport/ phosphorylation

1. Cyclic photophosphorylation:

• Light Absorption: Chlorophyll and other pigments in the thylakoid membrane absorb light energy from the sunlight.

• Electron Excitation: The absorbed light energy excites electrons in the chlorophyll molecules, raising them to higher energy levels.

• Electron Transport: Excited electrons are passed through a series of electron carriers embedded in the thylakoid membrane. As electrons move along this transport chain, they release energy.

• ATP Synthesis: The released energy is utilized to pump protons (H⁺) across the thylakoid membrane, establishing an electrochemical gradient. The accumulated protons create a potential energy difference  harnessed by ATP synthase embedded in the membrane. ATP synthase uses this energy to convert ADP (adenosine diphosphate) and inorganic phosphate (Pi) into ATP.

• Return of Electrons: After releasing energy, the electrons that were excited in step 2 return to the chlorophyll molecule from which they originated. This cyclic flow of electrons is characteristic of cyclic photophosphorylation.

2. Non-cyclic photophosphorylation:

• Non-cyclic photophosphorylation: a process that occurs during the light-dependent reactions of photosynthesis.

• It takes place in the thylakoid membrane of chloroplasts in plants, algae, and some bacteria.

• The primary goal of non-cyclic photophosphorylation is to produce both ATP (adenosine triphosphate) and NADPH (nicotinamide adenine dinucleotide phosphate).

• The process begins with the absorption of light energy by chlorophyll and other pigments in the thylakoid membrane.

• The absorbed light energy excites electrons in chlorophyll, raising them to higher energy levels.

• Excited electrons are transferred to electron carriers in a series of redox reactions, creating an electron transport chain.

• As electrons move along the electron transport chain, they release energy that is used to pump protons across the thylakoid membrane, establishing an electrochemical gradient.

• The accumulated protons create a potential energy difference harnessed by ATP synthase to produce ATP through the process of chemiosmosis.

• Simultaneously, during non-cyclic photophosphorylation, NADP⁺ (nicotinamide adenine dinucleotide phosphate) molecules accept the electrons at the end of the electron transport chain, becoming reduced to NADPH.

• NADPH, along with ATP, serves as a source of energy and reducing power for the subsequent light-independent reactions (Calvin cycle) of photosynthesis.

• The ultimate purpose of non-cyclic photophosphorylation is to capture and convert light energy into chemical energy in the form of ATP and NADPH, used as energy to synthesize carbohydrates and other organic compounds in the Calvin cycle.

Where are the ATP & NADPH used?

The Calvin cycle or C3 Pathway

• The Calvin cycle, also known as the light-independent reactions or dark reactions: is a series of biochemical reactions that occur in the stroma of chloroplasts in plants, algae, and some bacteria.

• Its primary function is to convert carbon dioxide (CO₂) into carbohydrates, particularly glucose, through a series of enzymatic reactions.

• The cycle consists of three main phases: carbon fixation, reduction, and regeneration of the CO2 acceptor molecule.

• Carbon Fixation:

  • CO₂ molecules from the atmosphere are captured and attached to a five-carbon sugar called ribulose-1,5-bisphosphate (RuBP).

  • This reaction is catalyzed by the enzyme RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase).

  • The resulting unstable six-carbon molecule splits into two three-carbon molecules called 3-phosphoglycerate (3-PGA).

• Reduction:

  • ATP and NADPH, generated during the light-dependent reactions, provide the energy and reducing power for the conversion of 3-PGA into glyceraldehyde-3-phosphate (G3P).

  • Some of the G3P molecules produced are used to regenerate RuBP, while others are utilized to synthesize glucose and other carbohydrates.

• Regeneration of the CO₂ Acceptor:

  • The remaining G3P molecules in the cycle undergo additional reactions to regenerate the initial CO₂ acceptor molecule, RuBP.

  • ATP is consumed in these reactions to rebuild the RuBP molecules, preparing them for another round of carbon fixation.

• The Calvin cycle is a cyclic process - some of the intermediate compounds are recycled to sustain the continuous production of carbohydrates.

• The cycle must occur multiple times to generate one glucose molecule, as six cycles are required to fix six molecules of CO₂.

• The Calvin cycle is crucial for the synthesis of organic molecules, including glucose. 

• The ATP and NADPH produced during the light reactions power the Calvin cycle's energy-demanding reactions.

C4 Pathway/ Hatch & Slack Pathway

• The C4 pathway is an alternative photosynthetic pathway that some plants have evolved to enhance carbon dioxide (CO₂) fixation, particularly in hot and dry environments.

• The C4 pathway is found in plants such as maize (corn), sugarcane, sorghum, and certain grasses.

• The CO₂ fixation step occurs in specialized cells called mesophyll cells, which are located near the surface of the leaf.

• In mesophyll cells, CO₂ combines with a three-carbon molecule, phosphoenolpyruvate (PEP), to form a four-carbon compound called oxaloacetate (OAA) in the presence of the enzyme PEP carboxylase.

• OAA, is converted to malate or aspartate and transported to bundle sheath cells deeper within the leaf.

• In the bundle sheath cells, CO₂ is released from the transported compounds and enters the Calvin cycle.

• The separation of CO₂ fixation and the Calvin cycle into different cells reduces the effects of photorespiration and increases the efficiency of carbon fixation, especially under high light and high-temperature conditions.

• Overall, the C4 pathway allows plants to minimize water loss

  • through reduced stomatal opening, 

  • increase photosynthetic efficiency, 

  • adapt to environments with high light, high temperature, and low CO₂ conditions.

CAM Pathway

• The CAM pathway is a specialized form of photosynthesis observed in certain plants, particularly succulents, that grow in arid or semi-arid environments.

• CAM plants: cacti, pineapple, and some orchids.

• The CAM pathway allows plants to conserve water:

  •  by opening their stomata

  • and conducting gas exchange during the night when temperatures are cooler and humidity is higher.

• During the night, CAM plants open their stomata and take in carbon dioxide, then convert into a four-carbon organic acid called malate. 

• The malate is stored in large vacuoles until the following day.

• During the day, the stored malate is broken down releasing CO₂ that enters the Calvin cycle for photosynthesis, which enables carbohydrate synthesis and the production of glucose.

• The CAM pathway allows plants to efficiently use CO₂ by storing it at night and releasing it during the day, avoiding excessive water loss through transpiration.

• This pathway maximizes water-use efficiency in arid environments where water availability is limited.

• CAM plants have adapted their leaf anatomy and physiology to carry out this pathway, including specialized cells and biochemical mechanisms that regulate the storage and release of CO2.

• The CAM pathway is an example of a plant's ability to adapt to water-stressed conditions and efficiently utilize available resources.