Enzymes and Metabolism

Thermodynamics in Biology

• First law: energy cannot be created or destroyed, only transformed
• Second law: every energy transfer increases entropy (disorder) in the universe
• Free energy ($G$): energy available to do work

$\Delta G = \Delta H - T\Delta S$

• Exergonic ($\Delta G < 0$): spontaneous, releases energy (e.g., cellular respiration)
• Endergonic ($\Delta G > 0$): non-spontaneous, requires energy input (e.g., photosynthesis)

ATP — Energy Currency

• ATP (adenosine triphosphate) couples exergonic and endergonic reactions
• ATP hydrolysis: $ATP \rightarrow ADP + P_i$ releases ~30.5 kJ/mol
• Energy coupling: exergonic ATP hydrolysis drives endergonic cellular work (mechanical, transport, chemical)
• ATP is regenerated by substrate-level phosphorylation and oxidative phosphorylation

• Enzymes are mostly proteins (some are RNA — ribozymes)
• They lower the activation energy ($E_a$) without changing the equilibrium or $\Delta G$
• They are not consumed and can be reused

How Enzymes Work

1. Substrate binds to the active site (specific, complementary shape)
2. Induced-fit model: active site changes shape slightly to better fit the substrate
3. Enzyme-substrate complex forms → transition state stabilised
4. Reaction occurs → products released → enzyme returns to original shape

Enzymes lower $E_a$ by:

• Orienting substrates correctly
• Straining substrate bonds
• Providing a favourable microenvironment (local pH/charge)
• Directly participating in the reaction (temporary covalent bonds)

Substrate Concentration

• Rate increases as [substrate] increases (more active sites occupied)
• At high [substrate]: all active sites occupied → $V_{max}$ (saturation)

Temperature

• Increasing temperature → more kinetic energy → faster collisions → increased rate
• Optimum temperature: maximum rate (human enzymes ~37°C)
• Above optimum: denaturation (weak bonds break, active site changes shape)

pH

• Each enzyme has an optimum pH (e.g., pepsin ~2, trypsin ~8)
• Extreme pH changes ionisation of R-groups → disrupts bonds → denaturation

Enzyme Concentration

• More enzyme = more active sites = faster rate (if substrate is in excess)

Competitive Inhibition

• Inhibitor resembles substrate, binds active site
• Competes with substrate — can be overcome by adding excess substrate
• Increases apparent $K_m$; $V_{max}$ unchanged

Non-competitive (Allosteric) Inhibition

• Inhibitor binds allosteric site (different from active site)
• Changes enzyme shape → active site distorted
• Cannot be overcome by adding substrate
• $K_m$ unchanged; $V_{max}$ decreased

Uncompetitive Inhibition

• Binds only to the enzyme-substrate complex
• Decreases both $K_m$ and $V_{max}$

Irreversible Inhibition

• Permanently modifies the enzyme (e.g., nerve agents, heavy metals)

• Enzymes with multiple subunits can have allosteric sites
• Allosteric activators: stabilise active form → increase activity
• Allosteric inhibitors: stabilise inactive form → decrease activity
• Cooperativity: substrate binding to one active site increases affinity of other sites (e.g., haemoglobin O₂ binding — sigmoidal curve)

Feedback (End-Product) Inhibition

• The final product of a metabolic pathway inhibits an early enzyme
• Prevents overproduction; efficient regulation
• Example: isoleucine inhibits threonine deaminase in its own synthesis pathway

• Catabolic pathways: break down molecules, release energy (e.g., cellular respiration)
• Anabolic pathways: build molecules, consume energy (e.g., protein synthesis)
• Pathways are regulated by controlling key enzymes (often the first step)
• Compartmentalisation: different pathways occur in different organelles (glycolysis in cytoplasm, Krebs in mitochondrial matrix)

Question: An enzyme has a $V_{max}$ of 100 μmol/min. In the presence of inhibitor X, $V_{max}$ drops to 60 μmol/min but $K_m$ is unchanged. What type of inhibition is this? Explain. (3 points)

Solution:

This is non-competitive inhibition. In non-competitive inhibition, the inhibitor binds to an allosteric site, changing the enzyme's shape and reducing its catalytic efficiency. The $V_{max}$ decreases (from 100 to 60) because some enzyme molecules are always inhibited regardless of substrate concentration. The $K_m$ is unchanged because the inhibitor does not affect substrate binding to the active site — the unaffected enzyme molecules still have the same affinity for the substrate.

• Enzymes are biological catalysts that lower activation energy without changing $\Delta G$ or equilibrium.
• Induced-fit model: active site moulds around the substrate for optimal catalysis.
• Rate affected by: temperature, pH, [substrate], [enzyme], inhibitors.
• Competitive inhibitors block the active site (overcome by excess substrate); non-competitive bind allosteric sites (not overcome).
• Allosteric regulation and feedback inhibition control metabolic pathways efficiently.
• ATP is the universal energy coupling molecule, regenerated by phosphorylation.