Enzymes and Bioenergetics Chapter Notes | Biology for BMAT (Section 2) PDF Download

Enzymes: Classification and Mode of Action

• Enzymes are biocatalysts that accelerate biochemical reactions both in vivo and in vitro without being altered.
• Highly specific to substrates, they significantly enhance reaction rates.
• Most enzymes are proteins, except for catalytic RNA molecules called ribozymes.
• Enzyme molecular weights range from 2000 to over one million Daltons, similar to proteins.
• Enzymatic activity depends on conformational structure and can be disrupted by denaturation.
• Many enzymes require cofactors for activity, which can be coenzymes (complex organic molecules) or metal ions (e.g., Fe²⁺, Mn²⁺, Zn²⁺, Mg²⁺).
• A holoenzyme is an enzyme with its cofactor; the protein component alone is an apoenzyme.
• Coenzymes, often derived from vitamins, transiently participate in catalysis by carrying specific functional groups (e.g., Biocytin from Biotin transfers CO₂, NAD from Niacin transfers hydride).
• Metal ion cofactors include Fe²⁺/Fe³⁺ (catalase, peroxidase), Cu²⁺ (cytochrome oxidase), Mg²⁺ (DNA polymerase), Zn²⁺ (carbonic anhydrase), and others.
• Prosthetic groups are coenzymes or metal ions tightly bound to the enzyme via covalent bonds.

Classification of Enzymes

• Oxidoreductases: Catalyze oxidation-reduction reactions (electron transfer).
• Transferases: Transfer functional groups.
• Hydrolases: Catalyze hydrolytic reactions (group transfer to water).
• Lyases: Add or remove groups to form double bonds.
• Isomerases: Transfer groups within molecules to form isomers.
• Ligases: Condense two molecules using ATP hydrolysis.
• Translocase: Transfer ions/molecules across membranes.
• Isozymes are multiple forms of an enzyme in the same species, tissue, or cell, catalyzing the same reaction but differing in amino acid composition and physicochemical properties (e.g., hexokinase has four forms, lactate dehydrogenase has five in humans).
• The enzyme active site is a small, defined pocket or cleft where the substrate binds, formed by portions of the polypeptide chain with a three-dimensional structure.
• Substrate binding at the active site involves non-covalent interactions like electrostatic, hydrogen bonds, Van der Waals forces, and hydrophobic interactions.

Fischer’s Lock and Key Model

• Proposed by Emil Fischer in 1894, it suggests that the enzyme’s active site is pre-shaped to fit the substrate precisely, like a key fitting a lock.
• Complementary structural features between enzyme and substrate allow the formation of an enzyme-substrate complex.

Koshland’s Induced Fit Model

• Proposed by Daniel Koshland in 1958, it posits that the active site is not rigid but flexible, undergoing conformational changes upon substrate binding to align catalytic groups.
• The substrate’s structure is complementary to the active site only in the enzyme-substrate complex, not in the free enzyme, resembling a hand fitting a glove.

Enzyme Specificity

• Group specificity: Enzymes act on several closely related substrates.
• Absolute specificity: Enzymes act on only one substrate.
• Stereospecificity: Enzymes act on one stereoisomer (e.g., D-amino acid oxidase oxidizes D-amino acids, not L-amino acids).
• Geometrical specificity: Enzymes differentiate between cis and trans forms (e.g., fumarase interconverts fumarate and malate).

Factors Affecting Enzyme Activity

Enzyme-catalyzed reaction rates are influenced by environmental conditions like temperature, pH, substrate concentration, and modulators.

Temperature:

• Reaction rate increases with temperature up to an optimum, then declines, forming a bell-shaped curve.
• Optimum temperature varies; most human enzymes peak at ~37°C, while some (e.g., Taq DNA polymerase) function at 100°C.
• High temperatures denature or degrade enzymes.

pH:

• Enzyme activity peaks at an optimum pH, forming a bell-shaped curve.
• Most enzymes in higher organisms function best at neutral pH (6-8), with exceptions like pepsin (pH 1-2), acid phosphatases (pH 4-5), and alkaline phosphatases (pH 10-11).
• Extreme pH values reduce or eliminate activity.

Substrate Concentration:

• Reaction rate increases with substrate concentration until enzyme saturation, after which further increases have no effect.
• At saturation, the enzyme works at its maximum rate, limited by enzyme availability.

Unit of Enzyme Activity

• The enzyme unit (U) is the amount of enzyme that converts 1 micromole of substrate to product per minute under standard conditions, adopted by I.U.B. in 1964.
• The katal (kat) is preferred, measuring 1 mole of substrate converted per second (1 kat = 6 × 10⁷ IU), as the minute is not an SI unit.

Specific Activity

Mechanism of Enzyme Action

• Enzymes affect reaction rates by lowering the activation energy (ΔGᴬ), not the free energy difference (ΔG) between reactants and products.
• ΔG determines reaction spontaneity, while ΔGᴬ determines the rate; enzymes cannot alter thermodynamic equilibrium.
• Substrates transition to products via a high-energy transition state; enzymes reduce the energy barrier (ΔGᴬ) to speed up reactions.

Kinetics of Enzyme-Catalysed Reaction

• During catalysis, the enzyme (E) binds substrate (S) to form an enzyme-substrate complex (ES), which converts to product (P) and free enzyme: E + S ⇌ ES → E + P.
• Leonor Michaelis and Maud Menten (1913) explained enzyme kinetics, noting the ES complex as a key intermediate.
• The Michaelis-Menten equation is: v₀ = (V_max [S]) / (K_m + [S]), where v₀ is initial velocity, V_max is maximum velocity, [S] is substrate concentration, and K_m is the Michaelis constant.
• Plotting v₀ against [S] yields a rectangular hyperbola.
• At low [S] (≪ K_m), v₀ is proportional to [S]; at high [S] (≫ K_m), v₀ equals V_max; when [S] = K_m, v₀ = V_max/2.
• K_m is the substrate concentration at half the maximum reaction rate.

Enzyme Inhibition

• Competitive inhibition: Inhibitor (I) resembles substrate (S), competing for the active site, forming EI or ES complexes but not ESI. Increases K_m (denoted K_m′) but does not affect V_max.
• Non-competitive inhibition: Inhibitor binds a different site, not competing with substrate, reducing functional enzyme and lowering V_max without affecting K_m.
• Uncompetitive inhibition: Inhibitor binds only to the ES complex, not free enzyme, altering both K_m and V_max.

Allosteric Enzymes

• Allosteric enzymes deviate from Michaelis-Menten kinetics, producing a sigmoidal curve when v₀ is plotted against [S].
• Each subunit has a regulatory site alongside the active site; regulatory molecules bind reversibly, altering substrate affinity.
• They are key regulators of metabolic pathways, unlike most enzymes that follow Michaelis-Menten kinetics.

Brief Introduction to Bioenergetics

• Bioenergetics studies energy transformation and use in living cells, governed by thermodynamics.
• Energy is released as reactions move from higher to lower energy levels, used to perform work.

The Laws of Thermodynamics

• Thermodynamics deals with energy changes in biochemical processes, focusing on direction, work, and external energy needs.
• First Law: Energy cannot be created or destroyed, only converted; total energy in the universe (system + surroundings) is constant.
• Equation: ΔE = E_B - E_A = Q - W, where ΔE is the change in internal energy, E_A and E_B are initial and final energies, Q is heat absorbed, and W is work done.
• Energy change depends only on initial and final states, not the transformation path.
• Second Law: The universe’s entropy (disorder) always increases; a process is spontaneous if the total entropy (system + surroundings) increases: S_system + S_surroundings > 0.
• Entropy measures randomness; biological systems maintain low entropy (high order) using chemical or light energy, but entropy increases post-death.
• A system’s entropy can decrease if the surroundings’ entropy increases sufficiently.
• First law focuses on energy transformation; second law addresses energy availability for work.

Combining the Two Laws

• Entropy is impractical for assessing biochemical reaction spontaneity due to measurement challenges.
• Free energy (ΔG) combines the first and second laws: ΔG = ΔH - TΔS, where ΔH is enthalpy change, T is absolute temperature, and ΔS is entropy change.
• ΔG represents available work; TΔS is the unusable energy component.
• In a closed system (exchanging energy, not matter), at constant temperature and pressure: ΔE = ΔH - PΔV, where PΔV is work done by volume change.

ATP: The Universal Currency of Free Energy

• Living organisms derive free energy from sunlight (photosynthetic) or food oxidation (chemotrophs).
• Free energy supports macromolecule synthesis, active transport, muscle contraction, and genetic information transfer.
• Energy is partly converted to adenosine triphosphate (ATP), the universal energy currency.
• During exergonic processes (e.g., food breakdown), ATP is synthesized from ADP and inorganic phosphate (Pi).
• ATP donates energy to endergonic processes (e.g., biosynthesis, transport), converting to ADP and Pi, releasing 7.3 kcal/mol.
• In some processes (e.g., firefly light production), ATP converts to AMP and pyrophosphate (PPi).
• ATP, ADP, and AMP are nucleotides with adenine, ribose, and three, two, or one phosphate group(s), respectively.