3.3.4 Substrate Concentration

Definition: The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximum rate (Vmax) is reached.

Key Points:

• At low substrate concentration, the rate of reaction is directly proportional to substrate concentration.
• As substrate concentration increases, the rate of reaction increases.
• At a certain point, the rate of reaction levels off and reaches a maximum.
• This maximum rate is called Vmax.
• At Vmax, all enzyme active sites are saturated with substrate.
• Further increase in substrate concentration will not increase the rate of reaction.
• The enzyme is said to be saturated.

Graph: - A hyperbolic curve is obtained when rate of reaction is plotted against substrate concentration.

Michaelis-Menten Equation: V₀ = V_max [S]K_m + [S]

• V₀ = Initial velocity
• V_max = Maximum velocity
• [S] = Substrate concentration
• K_m = Michaelis constant

• The substrate concentration at which the reaction rate is half of Vmax.
• Km is a measure of the affinity of the enzyme for the substrate.
• Low Km = high affinity (enzyme binds substrate tightly).
• High Km = low affinity (enzyme binds substrate loosely).

Intermediate Level

Temperature in Detail

• Rate increases with temperature up to optimum.
• Rate decreases sharply above optimum (denaturation).

pH in Detail

Graph: - Bell-shaped curve for each enzyme.

Enzyme Concentration in Detail

Substrate Concentration in Detail

Comparison of Factors

Important Points

• 1. The rate of enzymatic reaction is measured by the amount of substrate changed or amount of product formed, during a period of time.
• 2. The external conditions which affect rate of enzyme reactions are:
• - Temperature
• - pH
• - Concentration of enzyme
• - Substrate concentration
• 3. 3.3.1 Temperature:
• - Heating increases molecular motion.
• - Thus the molecules of the substrate and enzyme move more quickly, so probability of a reaction to occur is increased.
• - Increasing temperature affect the rate of reaction in such a way that an increase of just 10°C in the existing temperature doubles the rate of reaction but this effect remains up to a certain limit.
• - The temperature that promotes maximum activity is called an optimum temperature.
• - If the temperature is increased above this level, then a decrease in the rate of the reaction occurs despite the increasing frequencies of collision.
• - This is because the secondary and tertiary structures of the enzyme have been disrupted and the enzyme is said to be Denatured.
• - The enzyme unfolds and the precise structure of the active site is gradually lost.
• - This temperature which causes denaturation of enzyme is called maximum temperature.
• - The bonds which are most sensitive to temperature change are hydrogen bonds.
• - All human enzymes have a optimum temperature of about 37–38°C, but bacteria living in hot springs may have an optimum temperature of 70°C or higher.
• - Such enzymes have been used in biological washing powders for high temperature washes.
• - If temperature is reduced to near or below freezing point, enzymes are inactivated, not denatured.
• - They will regain their catalytic influence when higher temperatures are restored.
• - This temperature where an inactive enzyme becomes active again is called minimum temperature.
• 4. 3.3.2 pH:
• - Every enzyme functions most effectively over a particular pH range.
• - This narrow range of pH at which the maximum rate of reaction is achieved is called optimum pH.
• - Enzyme conformation is sensitive to pH changes because pH influences the charges on the amino acid side chains that are involved in maintaining tertiary and quaternary structure of enzyme.
• - Slight change in optimum pH of an enzyme causes ionization of amino acid of the enzyme therefore, they become inactive temporarily.
• - On the other hand, extreme changes in optimum pH alter the ionic charge of the acidic and basic groups of enzyme and the enzyme becomes denatured.
• - Optimum pH of different enzymes:
• - Pepsin – pH 2 (stomach)
• - Trypsin – pH 8 (small intestine)
• - Papain – pH 6–7 (papaya)

Remember

• - Temperature = increases rate up to optimum; denaturation above; inactivation below
• - Optimum temperature = maximum activity
• - **Human enzymes** = 37–38°C
• - Thermophilic bacteria = 70°C or higher
• - pH = each enzyme has an optimum pH
• - **Pepsin** = pH 2
• - **Trypsin** = pH 8
• - **Papain** = pH 6–7
• - Enzyme concentration = directly proportional (if substrate is not limiting)
• - Substrate concentration = increases rate up to Vmax; then saturates
• - Vmax = maximum rate
• - Km = substrate concentration at half Vmax
• - **Low Km** = high affinity
• - **High Km** = low affinity

Common Mistakes

• 1. Thinking enzymes are denatured at low temperature – Enzymes are inactivated at low temperature, not denatured.
• 2. Thinking enzymes work best at any pH – Each enzyme has a specific optimum pH.
• 3. Confusing optimum temperature and maximum temperature – Optimum = maximum activity; Maximum = denaturation.
• 4. Thinking enzyme concentration always increases rate – Only if substrate is not limiting.
• 5. Thinking substrate concentration always increases rate – Only up to Vmax; then saturates.
• 6. Confusing Km and Vmax – Km = substrate concentration at half Vmax; Vmax = maximum rate.
• 7. Thinking low Km means low affinity – Low Km = high affinity.
• 8. Thinking high Km means high affinity – High Km = low affinity.
• 9. Confusing competitive and non-competitive inhibitors – Competitive = binds active site; Non-competitive = binds allosteric site.
• 10. Thinking denaturation is reversible – Denaturation is usually irreversible.
• 11. Thinking pH affects only the substrate – pH affects the enzyme's amino acid side chains.
• 12. Forgetting the role of hydrogen bonds – Hydrogen bonds are most sensitive to temperature changes.

Quick Revision

• The rate of enzymatic reaction is measured by the amount of substrate changed or amount of product formed, during a period of time.
• The external conditions which affect rate of enzyme reactions are: temperature, pH, concentration of enzyme and substrate concentration.
• Temperature:
• Heating increases molecular motion.
• An increase of just 10°C in the existing temperature doubles the rate of reaction but this effect remains up to a certain limit.
• The temperature that promotes maximum activity is called an optimum temperature.
• If the temperature is increased above this level, then a decrease in the rate of the reaction occurs.
• This is because the secondary and tertiary structures of the enzyme have been disrupted and the enzyme is said to be Denatured.
• The temperature which causes denaturation of enzyme is called maximum temperature.
• The bonds which are most sensitive to temperature change are hydrogen bonds.
• All human enzymes have a optimum temperature of about 37–38°C, but bacteria living in hot springs may have an optimum temperature of 70°C or higher.
• If temperature is reduced to near or below freezing point, enzymes are inactivated, not denatured.
• This temperature where an inactive enzyme becomes active again is called minimum temperature.
• pH:
• Every enzyme functions most effectively over a particular pH range.
• This narrow range of pH at which the maximum rate of reaction is achieved is called optimum pH.
• Enzyme conformation is sensitive to pH changes because pH influences the charges on the amino acid side chains that are involved in maintaining tertiary and quaternary structure of enzyme.
• Slight change in optimum pH of an enzyme causes ionization of amino acid of the enzyme therefore, they become inactive temporarily.
• Extreme changes in optimum pH alter the ionic charge of the acidic and basic groups of enzyme and the enzyme becomes denatured.
• Optimum pH of different enzymes:
• Pepsin – pH 2
• Trypsin – pH 8
• Papain – pH 6–7

60-Second Revision

• Temperature = increases rate up to optimum; denaturation above; inactivation below
• Optimum temperature = maximum activity
• **Human enzymes** = 37–38°C
• Thermophilic bacteria = 70°C or higher
• pH = each enzyme has an optimum pH
• **Pepsin** = pH 2
• **Trypsin** = pH 8
• **Papain** = pH 6–7
• Enzyme concentration = directly proportional (if substrate is not limiting)
• **Substrate