Bioenergetics and Enzymes – Lecture 10 Notes

Energy Fundamentals

• Energy = capacity to do work or supply heat. It is a fundamental property of matter and can exist in various forms.

  • Two primary forms:

  • Potential Energy (stored) - Energy possessed by an object or system due to its position, composition, or state. It represents stored energy that can be converted into kinetic energy.

    • Depends on position (e.g., gravitational potential energy), chemical bonds (chemical potential energy), concentration gradients (e.g., solute concentration difference across a membrane), or charge imbalance (electrochemical potential energy).

    • E.g., energy stored in chemical bonds of complex molecules like \text{ATP}, the potential energy of water held behind a dam, or the electrochemical gradient of ions across a cell membrane.

  • Kinetic Energy (in motion) - Energy of motion. This is the energy that is actively doing work.

    • Manifested as heat (random molecular motion producing thermal energy), mechanical movement (e.g., muscle contraction), light (photons), or electricity (flow of electrons).

    • E.g., the movement of molecules, the contraction of muscles, or the flow of electrical current.

• Interconversion: Potential energy can be converted into kinetic energy and vice versa. Energy is neither created nor destroyed, only transformed (see Laws of Thermodynamics).

Thermodynamics

• Thermodynamics = study of energy transformations within matter (the "system") and its surroundings. A "system" can be anything from a single cell to an entire organism, or even a specific biochemical reaction mixture.

• 1st Law of Thermodynamics (Law of Conservation of Energy) - Energy cannot be created or destroyed, only transformed from one form to another or transferred between systems. The total energy in the universe remains constant.

  • Biological relevance: Cells constantly convert chemical energy from food (potential energy) into other forms like kinetic energy (for movement), thermal energy (heat), and chemical energy in \text{ATP}, but the total energy is conserved.

• 2nd Law of Thermodynamics (Law of Increased Entropy) - Every energy transformation or transfer increases the entropy (disorder or randomness) of the universe and/or releases unusable energy, often as heat, to the surroundings. Spontaneous processes always increase the total entropy of the universe.

  • Living systems maintain a high degree of internal order (low entropy) at the expense of increasing the total disorder (entropy) of their surroundings by releasing heat and simpler waste products. This process requires a continuous input of energy.

Gibbs Free Energy (G)

• Gibbs Free Energy = The portion of a system's total energy that is available to perform useful work when temperature and pressure are uniform, as in a living cell. It is a measure of a system's instability.

• Systems spontaneously move toward a state of lower G (more stable, less capacity for work) and higher entropy.

• The change in free energy for a chemical reaction: \Delta G = G{\text{products}} - G{\text{reactants}}. Another way to express it is \Delta G = \Delta H - T\Delta S where:

  • \Delta H (Enthalpy) = change in the system's total energy (heat content).

  • T = absolute temperature in Kelvin.

  • \Delta S (Entropy) = change in the system's disorder or randomness.

• Interpreting \Delta G:

  • \Delta G < 0 (negative): Energy is released from the system; the reaction is spontaneous (exergonic) and energetically favorable. It can proceed without continuous energy input.

  • \Delta G > 0 (positive): Energy is absorbed by the system; the reaction is non-spontaneous (endergonic) and requires an input of energy to proceed.

  • \Delta G = 0: The system is at equilibrium; no net change occurs.

Energy Transformation Examples

• Gravitational motion: A rock at the top of a hill has high positional potential energy (G). As it rolls down, this potential energy is converted into kinetic energy (motion and heat), leading to a state of lower positional G.

• Diffusion: Molecules concentrated in one area (high potential energy due to concentration gradient) spontaneously spread out to occupy the entire available volume (low potential energy, higher entropy) until evenly dispersed.

• Chemical reaction: The breaking of high-energy chemical bonds in reactants to form lower-energy products releases free energy, often as heat and useful work. For example, catabolic reactions break down complex molecules into simpler ones, releasing energy.

Exergonic vs Endergonic Reactions

• Exergonic ("exo" meaning outside + "gonic" meaning work) - Reactions that release free energy into the surroundings. They are energetically favorable processes.

  • Characteristic: \Delta G < 0. They are spontaneous processes.

  • Example: The hydrolysis of adenosine triphosphate (\text{ATP}) to adenosine diphosphate (\text{ADP}) and inorganic phosphate (\text{P}_\text{i}) is highly exergonic, releasing approximately -30.5\,\text{kJ·mol}^{-1} of energy.

• Endergonic ("endo" meaning inside + "gonic" meaning work) - Reactions that absorb free energy from the surroundings. They are energetically unfavorable processes.

  • Characteristic: \Delta G > 0. They are non-spontaneous and require an input of energy to occur.

  • Example: The synthesis of glucose-6-phosphate from glucose and inorganic phosphate is an endergonic reaction. In cells, endergonic reactions are often "coupled" to exergonic reactions (like ATP hydrolysis) to allow them to proceed.

Activation Energy (E_A)

• E_A = The minimum amount of energy that reactant molecules must absorb from their surroundings to reach the unstable "transition state," where bonds can be broken and new bonds can form. It determines the rate of a reaction but does not affect the overall \Delta G of the reaction.

• Even highly exergonic reactions require an initial input of E_A to start.

• Higher E_A means a slower reaction rate. Lowering E_A increases the reaction rate.

• Raising the temperature can increase the kinetic energy of molecules, helping them overcome E_A. However, this is not a viable widespread solution in biological systems because excessive heat can denature proteins (destroy their structure and function) and damage cell membranes.

Enzymes: Biological Catalysts

• Definition: Biological catalysts are macromolecular biological compounds (mostly proteins, but some RNA molecules called ribozymes) that accelerate the rate of specific biochemical reactions by lowering the activation energy (E_A) without being consumed in the process. They do not alter the overall \Delta G or the equilibrium position of a reaction; they only speed up the attainment of equilibrium.

• Common enzymes & functions:

  • Cellulase – Hydrolyzes cellulose into smaller sugars.

  • Kinase – Catalyzes the transfer of a phosphate group, typically from \text{ATP}, to a substrate (phosphorylation).

  • Phosphatase – Catalyzes the removal of a phosphate group from a substrate (dephosphorylation).

  • Lipase – Degrades lipids (fats) into fatty acids and glycerol.

  • Polymerase – Synthesizes polymers (e.g., DNA polymerase, RNA polymerase) via dehydration reactions.

  • Ribozyme – Catalytic RNA molecules, such as the peptidyl transferase activity of the ribosome during protein synthesis.

Structure & Specificity

• Active Site: A specific, three-dimensional groove or pocket on the enzyme molecule where substrates bind. It is typically formed by amino acid residues that come together from different parts of the protein's polypeptide chain when the protein folds into its specific tertiary structure.

• Substrate specificity: Enzymes are highly specific for their substrates, meaning each enzyme typically catalyzes only one or a small number of related reactions. This specificity is based on the precise complementary shape, charge distribution, and hydrophobic/hydrophilic interactions between the active site and the substrate.

  • "Lock-and-key" model: An older model suggesting a rigid fit between enzyme and substrate.

  • "Induced fit" model: A more accurate model where the active site is flexible and slightly changes its shape upon substrate binding to create an optimal fit, enhancing the catalytic efficiency. It's like a scientific handshake between the enzyme and substrate.

• Enzyme–substrate complex: The transient molecular association formed when the enzyme's active site binds to the substrate(s) during catalysis. This complex is crucial for the reaction to occur.

Mechanisms of E_A Reduction

Enzymes lower activation energy through various mechanisms:

• Proper substrate orientation: Enzymes orient multiple substrates correctly in the active site, increasing the probability of effective collisions between reactive groups.

• Straining/bending substrate bonds: The enzyme's induced fit can physically strain or bend specific bonds within the substrate molecules, making them easier to break and reducing the energy required for the transition state.

• Providing a favorable microenvironment: The active site can provide specific chemical conditions not found in the bulk solution, such as a localized acidic or basic environment, or the exclusion of water, which favors the reaction.

• Direct participation in the reaction: Amino acid residues in the active site can directly participate in the chemical reaction by temporarily forming covalent bonds with the substrate, or by acting as proton donors/acceptors, facilitating the reaction path.

Steps of Catalysis

1. Substrate Binding: The substrate(s) enters the active site of the enzyme and binds, forming the enzyme–substrate complex (ES).

2. Transition State Formation and Catalysis: The enzyme facilitates the conversion of substrate(s) to product(s) by lowering E_A. The substrate is converted into the unstable transition state, and then into the product(s); bonds are broken and/or formed.

3. Product Release: The product(s) detach from the active site. The enzyme is then regenerated in its original state, with its active site free to bind new substrate molecules and initiate another catalytic cycle.

Reaction Coordinate Diagram

• A graphical representation showing the energy changes during a reaction. Without an enzyme, the reaction pathway features a higher E_A peak (transition state). With an enzyme, the E_A peak is significantly lowered, facilitating a faster reaction rate. Crucially, the starting (reactant) and ending (product) free energies remain the same, meaning the \Delta G for the overall reaction is unaltered by the enzyme.

Factors Affecting Enzyme Activity

Enzyme activity is highly sensitive to environmental conditions and other regulatory molecules.

• Environment:

  • Temperature: Reaction rate generally increases with temperature up to an optimal point, as increased kinetic energy leads to more frequent collisions between enzyme and substrate. Beyond the optimal temperature, the enzyme rapidly denatures (loses its specific three-dimensional shape, particularly the active site, due to the breaking of weak bonds like hydrogen bonds and ionic interactions), causing a sharp decrease in activity. For most human enzymes, the optimum is around 37^ ext{o}\text{C}, while enzymes from extremophile organisms (e.g., thermophiles) can have much higher optima (e.g., 77^ ext{o}\text{C}).

  • pH: Each enzyme has an optimal pH range. Deviations from this optimum alter the ionization state of amino acid residues in the active site and other critical regions of the enzyme, as well as the substrate itself. This can disrupt substrate binding or catalysis, leading to denaturation and a significant decrease in enzyme activity. For example, pepsin (found in the stomach) has an acidic optimum pH of approximately 2, while trypsin (found in the intestine) functions optimally at a slightly alkaline pH of approximately 8.

  • Salinity: Extreme changes in salt concentration can affect the ionic bonds within the enzyme, altering its protein folding and compromising its structure and function. Most enzymes have a narrow optimal salinity range.

• Enzyme Concentration: Increasing the concentration of the enzyme will generally lead to a faster reaction rate, provided there is an ample supply of substrate. The rate increases linearly with enzyme concentration until the substrate becomes the limiting factor.

• Substrate Concentration: As substrate concentration increases, the reaction rate also increases because more active sites are occupied. However, at a certain point, the enzyme becomes "saturated," meaning all available active sites are continuously occupied by substrate molecules. At saturation, further increases in substrate concentration will not increase the reaction rate unless more enzyme is added.

• Inhibitors: Molecules that decrease an enzyme's activity.

  • Competitive inhibitors: These molecules resemble the enzyme's natural substrate and compete with the substrate for binding to the active site. Their effect can often be overcome by increasing the substrate concentration.

  • Noncompetitive (allosteric) inhibitors: These molecules bind to a site on the enzyme other than the active site (an "allosteric site"). This binding causes a conformational change in the enzyme, resulting in a distorted active site that is less effective or completely unable to catalyze the reaction. Increasing substrate concentration does not overcome noncompetitive inhibition.

• Phosphorylation / Dephosphorylation: A common mechanism of post-translational covalent modification that regulates enzyme activity. A phosphate group (usually from \text{ATP}) is added to specific amino acid residues (like serine, threonine, or tyrosine) by an enzyme called a kinase. This phosphorylation can induce a conformational change in the enzyme, activating or inactivating it. Conversely, a phosphatase enzyme removes the phosphate group (dephosphorylation), often reversing the effect. This process allows for rapid on/off switching of enzyme activity in response to cellular signals. The hydrolysis of \text{ATP} associated with phosphorylation releases a significant amount of energy (\Delta G \approx -30.5\,\text{kJ·mol}^{-1}).

Example: Sucrose → Glucose + Fructose via Sucrase

• Consider the enzyme sucrase, which hydrolyzes sucrose into glucose and fructose. Here are scenario outcomes based on varying conditions:

  1. Add more enzyme: If substrate is not limiting, adding more sucrase will increase the number of available active sites, leading to an increased reaction rate until the substrate becomes the limiting factor.

  2. Heat moderately to optimum: Increasing the temperature up to the enzyme's optimum (e.g., 37^ ext{o}\text{C}) will increase the kinetic energy of molecules, leading to more frequent and effective collisions between sucrase and sucrose, thus increasing the reaction rate. However, excessive heat will cause denaturation of sucrase, leading to a sharp decrease in the rate.

  3. Cool solution: Decreasing the temperature will reduce the kinetic energy of molecules, resulting in fewer collisions between sucrase and sucrose, thereby decreasing the reaction rate.

  4. Decrease pH (acidify) away from optimum \approx neutral: Sucrase has an optimal pH around neutral. Decreasing the pH (making it more acidic) will alter the charge of amino acid residues in the active site and potentially denature the enzyme, reducing or eliminating its activity.

  5. Increase pH (alkalinize) away from optimum: Similarly, increasing the pH (making it more alkaline) will also alter the enzyme's charge distribution and conformation, leading to a decrease in reaction rate due to impaired active site function or denaturation.

  6. Add more sucrose: Increasing the substrate (sucrose) concentration will increase the reaction rate until all active sites on the sucrase molecules are saturated. At saturation, the rate will plateau, and adding more sucrose will not further increase the rate unless more enzyme is introduced.

Enzyme Inhibition & Regulation

Enzyme activity is tightly regulated within cells to control metabolic pathways.

• Competitive Inhibition:

  • Mechanism: The inhibitor molecule, which often structurally resembles the natural substrate, binds directly to the active site and competes with the substrate for binding. This blocks the substrate from binding.

  • Kinetic effects: The maximum reaction rate (V_{max}) of the enzyme generally remains unchanged (it can still reach V_{max} if enough substrate is added to outcompete the inhibitor), but the apparent Michaelis constant (K_m) increases. K_m is the substrate concentration at which the reaction rate is half V_{max}; a higher K_m indicates lower apparent affinity of the enzyme for its substrate because more substrate is needed to achieve half the maximum rate.

• Noncompetitive (Allosteric) Inhibition:

  • Mechanism: The inhibitor binds to a site on the enzyme that is distinct from the active site (an allosteric site). Binding at this site induces a conformational change in the enzyme, which in turn alters the shape of the active site, making it less effective at converting substrate to product.

  • Kinetic effects: The V_{max} of the enzyme decreases because fewer functional active sites are available regardless of how much substrate is present. The K_m typically remains unchanged because the inhibitor does not affect the actual binding affinity of the active sites that are still functional.

• Allosteric Activation:

  • Mechanism: An activator molecule binds to an allosteric site, stabilizing the enzyme in its active conformation. This can enhance substrate binding or catalytic efficiency.

  • Example: In some metabolic pathways, a product from a later step (e.g., Product D in an example pathway) might allosterically stimulate an enzyme earlier in the pathway (e.g., Enzyme 2), speeding up the production of intermediates required for its own synthesis.

• Feedback Inhibition (End-Product Inhibition):

  • Mechanism: A crucial regulatory mechanism where the final product of a metabolic pathway acts as a noncompetitive (allosteric) inhibitor of an enzyme earlier in the same pathway, often the first committed enzyme (the enzyme catalyzing the first irreversible step in the pathway).

  • Purpose: This prevents the wasteful overproduction of the end product and ensures that the pathway operates efficiently, maintaining cellular homeostasis by adjusting production based on demand.

  • Example: The amino acid isoleucine inhibits threonine deaminase, an enzyme involved in the first step of isoleucine synthesis.

• Feedback Activation (Positive Feedback):

  • Mechanism: A product of a pathway stimulates an earlier step in the same pathway, thus amplifying the pathway's activity.

  • Example: During childbirth, the hormone oxytocin stimulates uterine contractions, and these contractions, in turn, signal for the release of more oxytocin, creating a physiological positive feedback loop that intensifies labor.

Metabolism Overview

• Metabolism = The totality of an organism’s chemical reactions. It encompasses all processes of building up and breaking down molecules required for life.

• Metabolic Pathway: A series of sequential enzymatic steps that convert a specific starting molecule (substrate) into a final product. Each step is catalyzed by a specific enzyme (e.g., ext{A} \xrightarrow{\text{Enzyme 1}} \text{B} \xrightarrow{\text{Enzyme 2}} \text{C} \xrightarrow{\text{Enzyme 3}} \text{D}).

• Anabolic Pathways (Biosynthetic pathways):

  • Function: Build complex molecules from simpler ones.

  • Energy requirement: They consume energy; generally endergonic reactions (\Delta G > 0).

  • Examples: Protein synthesis (linking amino acids to form proteins), photosynthetic Calvin cycle (synthesizing glucose from \text{CO}_2), and DNA synthesis.

• Catabolic Pathways (Degradative pathways):

  • Function: Break down complex molecules into simpler ones.

  • Energy release: They release energy that can be captured and used to power anabolic reactions; generally exergonic reactions (\Delta G < 0).

  • Examples: Cellular respiration (breaking down glucose to produce \text{ATP}), digestion of food into monomers, and hydrolysis reactions.

• ATP's role: \text{ATP} (adenosine triphosphate) serves as the primary energy currency of the cell, linking catabolic (energy-releasing) reactions to anabolic (energy-requiring) reactions thro