Biochem Lecture Notes: Thermodynamics, Enzymes, Transition State, and Inhibitors

Thermodynamics and Coupled Reactions

• A dissociation example: one molecule splits into two; conceptual note on disorder (chaos) increasing with fragmentation.
• Negative numbers and arithmetic concept: negative minus a number remains negative.
• Inorganic phosphate vs organic phosphate:
  • Inorganic phosphate (Pi) is the phosphate group not attached to carbon.
  • If a phosphate is attached to carbon, it is considered organic phosphate; inorganic phosphate is the non-carbon-bound form.
• Negative ΔG indicates spontaneity and exergonic energy release. It is typically associated with catabolic processes.
• Coupling concept: reactions that are endergonic (not spontaneous) can be driven forward by pairing with a favorable exergonic reaction.
• Example setup: add up ΔG values to see if a coupled process is overall favorable.

Example: ATP hydrolysis and glucose phosphorylation

• ATP hydrolysis reaction:
  • ATP → ADP + inorganic phosphate (Pi)
  • ΔG = $ext{-7 kcal/mol}$ (negative, exergonic)
  • This energy release can drive other reactions forward.
• Glucose phosphorylation (adding Pi to glucose) is endergonic:
  • Glucose + Pi → glucose-6-phosphate (G6P)
  • ΔG = $+3.3 ext{ kcal/mol}$ (positive, non-spontaneous)
• Coupled reaction result:
  • Combine ATP hydrolysis with glucose phosphorylation to get:
  • Overall ΔG = $ext{-7} + ext{(+3.3)} = ext{-3.7 kcal/mol}$
  • Net result is exergonic (negative ΔG), so the coupled process proceeds spontaneously.
• Net equation after removing common species (balancing understanding):
  • Original components include water and Pi on both sides. Because these appear on both sides, they cancel, yielding:
  • $ext{ATP} <br /> ightarrow ext{ADP} + ext{glucose-6-phosphate}$
• Related thermodynamic values mentioned:
  • Hydrolysis of glucose-6-phosphate to glucose + Pi has ΔG ≈ $ext{-3.3 kcal/mol}$ (exergonic when considered alone in that direction).
  • Glucose-6-phosphate formation from glucose and Pi is endergonic with ΔG ≈ $+3.3 ext{ kcal/mol}$; coupling with ATP hydrolysis makes the coupled step exergonic with ΔG ≈ $-3.7 ext{ kcal/mol}$.
• Conceptual takeaway: cells often couple endergonic steps to exergonic ATP hydrolysis to make the overall process energetically favorable.
• Intuition about energy magnitudes: smaller magnitude ΔG in coupled steps allows rapid turnover and easier regulation; very large negative ΔG would require large energy input to reverse or renew energy currency.
• A brief note on energy currency strategy: easier to make and break small energy transactions (like relatively modest negative ΔG steps) than to rely on large negative steps that are harder to sustain or reverse in metabolism.

Enzymes and how they work

• Enzymes increase the rate of reactions (catalysis) and determine which reactions proceed in a cell by enabling endergonic steps to occur via coupling.
• Enzymes are not consumed in reactions; they act as catalysts that speed up the process without being used up.
• Key concept: transition state
  • During a reaction, there is a high-energy, unstable transition state between reactants and products. This state is short-lived and difficult to reach.
  • The transition state is a moment where bonds are partially broken/forming; it’s an unstable arrangement that determines the reaction rate.
• Activation energy (ΔG‡): the extra energy required to reach the transition state from reactants.
  • Enzymes work by stabilizing or binding the transition state, thereby lowering the activation energy needed to reach that state.
  • When an enzyme binds the transition state, it reduces ΔG‡, making the forward reaction more likely without altering the overall ΔG of the reaction.
• Important distinctions:
  • Enzymes do not change the Gibbs free energy change (ΔG) between reactants and products; they lower the activation barrier to speed up the reaction.
  • The catalyzed reaction has the same ΔG as the uncatalyzed one; only the activation energy is reduced.
• How the enzyme works in practice:
  • The enzyme binds the substrate in the active site to form the enzyme–substrate complex (ES).
  • The enzyme stabilizes the transition state; from there, the reaction can proceed to products or revert to reactants.
  • If products form, the product is released and the enzyme is free to catalyze again; the enzyme itself is unchanged after the reaction.
• Conceptual model for enzyme action:
  • E + S ⇌ ES → E + P
  • The transition state is the favored binding target for the enzyme; binding to the transition state lowers ΔG‡ and accelerates the reaction.
• Substrate vs reactant terminology:
  • In enzymology, the initial molecule is often called the substrate; the intermediate complex is ES; the end molecule is the product P. The terms “substrate” and “reactant” can be used interchangeably in some contexts, but “substrate” emphasizes enzyme binding.
• Active site and specificity:
  • The active site is formed by the folded three-dimensional structure of the enzyme, where specific amino acid side chains (R groups) create a pocket that binds the transition state of the substrate.
  • Mutations in active-site residues can greatly affect binding and catalysis; mutations elsewhere may have little effect if they don’t alter the active site.
  • The specificity of the active site ensures the enzyme catalyzes only its intended reaction and not others.
• Protein folding relevance:
  • Proper folding creates the active site; misfolding or mutations can disrupt the pocket and render the enzyme nonfunctional or less efficient.
• Practical lab analogies mentioned:
  • A demonstration concept with a semipermeable membrane and enzymes on one side to illustrate substrate binding and reaction localization; emphasis on whether substrates can cross membranes and how enzymes act on bound substrates.
• Summary takeaway about enzyme action:
  • Enzymes lower activation energy by binding the transition state, enabling reactions to proceed more readily without changing the overall energy balance of reactants vs products.

Inhibitors of enzymes

• Irreversible inhibitors
  • Bind to the enzyme and stay attached permanently.
  • Result: the enzyme is inactivated and cannot catalyze the reaction; the rate of reaction effectively drops to zero (or very slow if turnover is extremely slow).
• Reversible inhibitors
  • Include competitive and noncompetitive inhibitors (all reversible forms).
  • Competitive inhibitors
  • Bind to the active site, competing with the substrate for binding the enzyme.
  • When a competitive inhibitor is bound, the substrate cannot bind and the reaction rate decreases.
  • The inhibition can be overcome by increasing substrate concentration, which increases the chance the substrate binds the active site instead of the inhibitor.
  • Noncompetitive inhibitors (not detailed fully in the transcript but mentioned as a category of reversible inhibitors)
  • Bind to a site other than the active site, altering enzyme conformation and reducing activity without directly competing with substrate binding.
• Conceptual visualization used in teaching:
  • The “box on the table” analogy for transition-state binding and how inhibitors can block access to the critical transition-state geometry.
  • The idea that an enzyme must be able to release products and not bind products too tightly; otherwise, turnover would be hindered.
• Consequences for kinetics and regulation:
  • Inhibitors modulate the rate of catalysis by altering how readily the enzyme-substrate complex reaches the transition state and converts to product.
  • The presence of inhibitors can shift apparent Km (in competitive inhibition) or reduce Vmax (in noncompetitive inhibition), depending on the mechanism.
• Practical notes on enzyme inhibition and drug design:
  • Irreversible inhibitors are often potent blockers of enzyme activity; reversible inhibitors provide a controllable way to modulate activity.
  • Specificity of inhibitors to particular enzymes is crucial for targeted regulation with minimal off-target effects.

Key concepts to remember (summary checklist)

• ΔG exergonic vs endergonic:
  • If riangle G < 0, the process is exergonic and spontaneous.
  • If riangle G > 0, the process is endergonic and non-spontaneous.
• Coupling to drive endergonic reactions:
  • Coupling an endergonic step to a highly exergonic step (e.g., ATP hydrolysis) can render the overall process spontaneous.
• ATP hydrolysis energy benchmark:
  • $riangle G_{ ext{ATP hydrolysis}} ext{(in this context)} = -7 ext{ kcal/mol}$
• Glucose phosphorylation energetics:
  • $riangle G_{ ext{glucose + Pi → glucose-6-P}} = +3.3 ext{ kcal/mol}$
  • Net coupled process with ATP hydrolysis: $riangle G_{ ext{net}} = -7 + 3.3 = -3.7 ext{ kcal/mol}$
• Hydrolysis of glucose-6-phosphate: $riangle G_{ ext{G6P → glucose + Pi}} = -3.3 ext{ kcal/mol}$
• Activation energy and transition state:
  • Enzymes lower the activation energy $riangle G^<br /> eq$ by stabilizing the transition state, not by changing the overall $riangle G$ of reactants to products.
• Enzyme-substrate interactions:
  • Enzyme binds substrate to form ES; stabilizes the transition state; converts to product; enzyme is regenerated.
• Active site and mutational impact:
  • The active site is formed by the folded protein; mutations in active-site residues can drastically alter function by changing transition-state binding.
• Inhibitors and enzyme kinetics:
  • Irreversible inhibitors permanently deactivate enzymes.
  • Competitive inhibitors block the active site and can be overcome by high substrate concentration.
  • Noncompetitive inhibitors alter enzyme activity by binding away from the active site (reversible case).
• Terminology:
  • Substrate is often used for the molecule that binds the enzyme; after binding and reaction, it becomes product.
  • Enzymes are not consumed in the reaction; they facilitate turnover of many substrate molecules.

Real-world relevance and connections

• Understanding energy coupling is fundamental to metabolism (e.g., how cells drive endergonic biosynthetic reactions using the energy from ATP hydrolysis).
• Enzyme kinetics and transition-state theory explain how catalysts accelerate life-sustaining reactions and how inhibitors regulate metabolic pathways.
• The active site concept links protein folding to function: the three-dimensional arrangement determines specificity and catalytic efficiency.
• Inhibitors are central to pharmacology and drug design (e.g., targeting specific enzymes in pathogens or cancer cells while minimizing off-target effects).