Enzymes: Key Concepts, Kinetics, and Regulation

Enzyme classification (six major classes)

• Enzymes are proteins that catalyse specific biochemical reactions; they are classified into six major classes based on the type of reaction they catalyse. Each class has representative enzymes and characteristic reaction types:
  • Class 1: Oxidoreductase — catalyse redox reactions (transfer of H, O, or e−). Example: Alcohol dehydrogenase.
  • Class 2: Transferase — transfer a functional group from one molecule to another. Example: Hexokinase.
  • Class 3: Hydrolase — hydrolyse (break bonds) in the presence of water. Example: Glucose-6-phosphatase.
  • Class 4: Lyase — remove or add groups to a molecule (breaks bonds, electron rearrangement). Example: Pyruvate decarboxylase.
  • Class 5: Isomerase — rearrange functional groups in a molecule (isomers). Example: Phosphoglucose isomerase.
  • Class 6: Ligase — join molecules. Example: Pyruvate carboxylase.
• Redox terminology:
  • Electron transfer concepts: increased oxidation state = loss of electrons, reduced species gain electrons. Redox is often coupled to NAD+/NADH as electron carriers.

Enzyme nomenclature

• Suffix “ase” is used to name enzymes.
• Names are based on what the enzyme reacts with and how it acts; often include substrate name and type of reaction.
  • Example: Pyruvate decarboxylase — removes a carboxyl group from pyruvate.
  • Resulting products: acetaldehyde and CO₂ (in fermentation context).
• The enzyme name provides a concise descriptor of its catalytic activity.

Enzyme properties

• Key properties of enzymes:
  • Catalyst: speeds up reactions; lower activation energy but do not change the ultimate equilibrium.
  • Equilibrium attainment: reactions reach equilibrium more rapidly with enzymes but Keq remains unchanged by the enzyme presence.
  • Stability: enzymes retain their active conformations during reactions.
  • Specificity: enzymes catalyse a single reaction (or a group of closely related reactions).
  • Substrate specificity: enzymes interact with particular substrates.
  • Regulation: tight regulation in the cell to prevent chaotic activity; enzymes are controlled by multiple mechanisms.
• Substrate: the molecule(s) that binds to the enzyme’s active site to undergo a chemical transformation.

Enzyme mechanism and kinetics (basic framework)

• Enzyme-catalysed reaction scheme:
  • E + S ⇌ ES → E + P
  • The enzyme is not consumed; it is recycled after product formation.
• Active site and substrate binding:
  • Substrate binds at the enzyme’s active site.
  • Induced fit model: enzyme undergoes a conformational change upon substrate binding to better facilitate the chemical reaction and stabilize the transition state.
• The rate and outcome depend on multiple factors that govern catalysis and turnover.

Activation energy and catalysis

• Activation energy (E_A): the energy required to reach the transition state from reactants.
• Transition state: a high-energy, short-lived state that must be achieved for reaction to proceed.
• Ways to reach transition state more readily:
  • Add a catalyst (e.g., an enzyme) to lower the activation energy barrier.
  • Increase temperature to raise kinetic energy and collision frequency.
• Conceptual illustration: catalysts lower the energy barrier, allowing more molecules to reach transition state and form products.

Enzymes as catalysts (mechanism in detail)

• Enzyme–substrate complex formation:
  • E + S ⇌ ES
  • ES undergoes chemical transformation to form product: ES → E + P
• Induced fit and transition state stabilization:
  • Binding induces conformational changes that stabilise the intermediate and transition state, accelerating the reaction rate.
• The enzyme is not consumed; it can catalyse multiple substrate turnover cycles.

Factors affecting enzyme activity

• Key variables:
  • Enzyme concentration [E]
  • Substrate concentration [S]
  • Temperature
  • pH
• These factors influence the rate at which enzymes catalyse reactions and can modulate enzyme activity dramatically.

Enzyme concentration [E]

• As [E] increases, reaction velocity increases linearly (positive linear relationship) when [S] is in excess.
• Vmax is proportional to the amount of enzyme present:
  • $V<em>{max} = k</em>3 [E]$
  • Here, k3 is a reaction-rate constant (turnover-related parameter).

Substrate concentration [S]

• As [S] increases, reaction velocity increases until a maximum is reached (Vmax).
• Low [S]: velocity increases roughly linearly with [S].
• Km: the substrate concentration at which the reaction velocity is half of Vmax.
• High [S]: velocity approaches Vmax and becomes independent of [S].
• Michaelis-Menten framework leads to the relationship:
  • v = rac{V_{max}",[S]
  • 
    }{K_m + [S]}
• Figure reference: Michaelis–Menten curve illustrating saturation of enzyme at high [S].

Why Km and Vmax are important

• Vmax: maximum rate when all enzyme active sites are saturated with substrate.
• Km: substrate concentration at half-maximum velocity; reflects the enzyme’s affinity for its substrate.
• Km has an inverse relationship with substrate binding affinity: lower Km indicates higher affinity.
• These parameters allow prediction of reaction rates under different substrate conditions.

Temperature effects

• Temperature influences enzyme activity:
  • At low temperatures, enzymes are largely inactive due to slow molecular movements.
  • As temperature rises, reaction velocity increases due to higher kinetic energy and collision frequency.
  • Extreme temperatures denature proteins by disrupting non-covalent interactions, reducing activity.
• Typical values:
  • Human enzymes often have peak activity near 37°C (body temperature).
  • Thermophilic bacterial enzymes may peak around ~75°C.
  • Above optimal temperature, rapid denaturation occurs.

pH effects

• Enzymes have activity within specific pH ranges (about 3–4 pH units wide for many enzymes).
• Extreme pH values disrupt electrostatic bonds and can denature enzymes.
• Specific examples of optimal pH:
  • Pepsin (stomach) ~pH 2.0
  • Trypsin (intestine) ~pH 8.0
• pH affects ionisation of amino and carboxyl groups, altering catalytic activity and stability.

Enzyme regulation

• Substrate-level regulation: regulation that affects substrate/products and enzyme interactions, but is not sufficient alone to control all cellular processes.
• Allosteric regulation: regulation via sites other than the active site that modulate activity.
  • Allosteric inhibition: reduces enzyme activity via conformational changes.
  • Allosteric activation: increases enzyme activity via conformational changes.
• Feedback inhibition: end products inhibit earlier steps to regulate pathway flux.

Covalent modification and proteolytic regulation

• Covalent modification: addition or removal of chemical groups (e.g., phosphorylation/dephosphorylation).
• Proteolytic cleavage: removal of a peptide segment to activate or inactivate enzymes (proteins synthesized as inactive zymogens).
  • Example: Enterokinase cleaves a hexapeptide from trypsinogen to activate trypsin.
• These regulatory mechanisms provide rapid and reversible or irreversible control of enzyme activity.

Glycogen phosphorylase as a model of regulatory control

• Phosphorylation/Dephosphorylation cycle:
  • Phosphorylation by phosphorylase kinase converts inactive glycogen phosphorylase b to active glycogen phosphorylase a.
  • Dephosphorylation by phosphorylase phosphatase inactivates glycogen phosphorylase a.
  • This cascade demonstrates covalent modification as a major control point in metabolism.

Textbook readings and sources

• Chapter 6 Enzymes: The catalysts of life (textbook reference)
• Becker’s World of the Cell by Hardin, Jeff (9th edition, 2018) and related figures (6-1, 6-9, 6-8, 6-4, 6-13, 6-14) cited in the slide deck.
• Additional reading: https://ebookcentral.proquest.com/lib/murdoch/detail.action?docID=5177602

Equilibrium concepts and examples

• Equilibrium constant Keq measures directionality of a reversible reaction and is defined as:
  • $K_{eq} = \frac{[B]}{[A]}$
• Keq is a property of the chemical system itself and remains the same regardless of enzyme presence.
• Example (hypothetical data): the conversion between glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) can be described with Keq = [F6P]/[G6P]. A hypothetical table (purportedly) might show:
  • For concentrations [F6P] = 6 mM and [G6P] = 12 mM, Keq = 0.5.
  • For [F6P] = 9 mM and [G6P] = 12 mM, Keq = 0.75.
  • For [F6P] = 3 mM and [G6P] = 12 mM, Keq = 0.25.
• Note: These numbers are hypothetical illustrative values; actual Keq depends on the specific reaction conditions.

Michaelis–Menten kinetics in detail

• Key terms:
  • Vmax: maximum velocity when enzymes are saturated with substrate.
  • Km: substrate concentration at which reaction velocity is half of Vmax.
• Michaelis–Menten equation:
  • $v = \frac{V<em>{max} [S]}{K</em>m + [S]}$
• Relationship to affinity:
  • Km inversely relates to binding affinity: lower Km indicates higher affinity between enzyme and substrate.
• High [S] regime:
  • When [S] >> Km, velocity approaches Vmax and becomes independent of [S].

Practical implications for experiments and interpretation

• To increase reaction rate in vitro, increasing enzyme concentration or substrate concentration within reason can raise velocity up to saturation.
• Understanding Km helps predict how changes in substrate concentration affect reaction rate in vivo and in assays.
• Temperature and pH optimization is critical for enzymatic assays; deviations can lead to misinterpretation of enzyme activity.
• Regulation (allosteric, covalent, proteolytic) provides multiple layers of control in metabolic pathways, allowing fine-tuning of flux.