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Enzyme Activity & Properties

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Properties of Enzymes

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Factors Affecting Enzyme Activity

1. Substrate Concentration — The Michaelis-Menten Relationship

v = Vmax · [S] / (KM + [S])

• Vmax = maximum velocity achieved when all enzyme active sites are saturated with substrate
• KM (Michaelis constant) = the substrate concentration at which the reaction rate is half of Vmax; it reflects the affinity of the enzyme for its substrate
  • Low KM → high affinity (enzyme is half-saturated at a low [S])
  • High KM → low affinity
• At very low [S]: reaction is first-order (rate proportional to [S])
• At saturating [S]: reaction is zero-order (rate independent of [S])

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2. Temperature

• Every 10°C rise approximately doubles enzyme activity (Q₁₀ ≈ 2)
• Higher temperatures improve sensitivity and speed — useful when enzyme activity is low
• Lower temperatures extend the linear range of assays, reducing the need for dilutions
• Upper limit: most enzymes begin to denature as temperature rises beyond their stability threshold
  • CK begins to denature at 37°C
  • Amylase begins to denature at 45°C
  • Exception: Taq polymerase (used in PCR) is stable at 95°C
• For accuracy, reaction temperature must not deviate more than ±0.1°C from the assigned value
• Cold storage effect: some enzymes are unstable at low temperatures (e.g., LD isoenzymes 4 and 5 lose activity at 4°C; CK-MB is stable at −20°C for months)

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3. pH

• Each enzyme has an optimal pH at which activity is maximal
• Deviations from the optimum reduce activity by:
  • Altering the ionization state of amino acid residues at the active site
  • Changing enzyme conformation
  • Affecting substrate binding
• Examples:
  • Alkaline phosphatase (ALP): optimum pH ~9
  • Acid phosphatase (ACP): optimum pH ~5
• In clinical assays, buffers maintain a stable pH to ensure reproducibility

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4. Enzyme Concentration

• At saturating substrate concentrations, reaction velocity is directly proportional to enzyme concentration — the basis for measuring enzyme activity in serum
• Enzyme levels depend on their rates of synthesis (transcriptional and translational control) and degradation
• Presence of substrate or inducing molecules can cause sudden increases in enzyme levels

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5. Inhibition of Enzymes

a) Competitive Inhibition

• The inhibitor structurally resembles the substrate and competes for the active site
• Increases the apparent KM (reduces affinity) but does not change Vmax
• Overcome by increasing substrate concentration
• Example: pyruvate acting as a competitive inhibitor of LD in reverse reactions

b) Noncompetitive Inhibition

• Inhibitor binds to a site other than the active site (an allosteric site)
• Binds equally to free enzyme and enzyme–substrate complex
• Vmax is reduced; KM is unchanged
• Cannot be overcome by increasing substrate

c) Uncompetitive Inhibition

• Inhibitor binds only to the enzyme–substrate complex
• Both Vmax and KM decrease (apparent KM decreases)

d) Irreversible Inhibition

• Inhibitor forms a covalent bond with the enzyme, permanently inactivating it
• Example: organophosphates irreversibly inhibit acetylcholinesterase

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6. Activators and Cofactors

• Many enzymes require cofactors (non-protein components) for activity:
  • Metal ions (Mg²⁺, Zn²⁺, Ca²⁺, Cl⁻) act as activators
  • Coenzymes (e.g., NAD⁺, NADP⁺, FAD) are organic cofactors, often vitamin-derived
• Removal of these cofactors renders the enzyme inactive (the protein alone = apoenzyme; enzyme + cofactor = holoenzyme)
• Specific ions can act as activators or inhibitors depending on concentration

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7. Allosteric Regulation

• Binding of small regulatory molecules to allosteric sites causes conformational changes
• Can either activate or inhibit the enzyme
• Enzymes subject to allosteric regulation typically catalyze rate-determining steps in metabolic pathways
• Allosteric enzymes show sigmoidal (not hyperbolic) kinetics — they display cooperativity

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8. Isoenzymes (Isozymes)

• Multiple molecular forms of the same enzyme with different biochemical properties (different pH optima, thermal stability, kinetics, tissue distribution)
• Different isoforms are expressed in different organs, making them useful tissue-specific biomarkers
• Example: LD has 5 isoenzymes; CK has 3 (CK-MM, CK-MB, CK-BB)

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9. Product Inhibition

• As the reaction proceeds and products accumulate, they can act as end-product inhibitors
• This is why kinetic assays measure initial velocity (before significant product builds up)
• Example: pyruvate accumulation in LD assays inhibits the reaction and can reverse it

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Summary Table

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