Factors affecting enzyme action

· Enzyme-catalysed reaction rate is affected by temperature, pH, enzyme concentration, substrate concentration and inhibitor concentration.
· In enzyme investigations, measure initial rate where possible, because substrate concentration has not yet fallen significantly.
· Rate can be measured by product formation or substrate disappearance.
· Keep all other variables constant so only the chosen independent variable affects the rate.
· Use repeats, calculate a mean, identify anomalies and plot suitable graphs to interpret trends.

Temperature

· As temperature increases, enzyme and substrate molecules gain kinetic energy.
· This causes more frequent successful collisions, so rate increases up to the optimum temperature.
· Above the optimum, bonds maintaining the enzyme’s tertiary structure break.
· The active site changes shape, so fewer enzyme–substrate complexes form.
· At high temperatures, the enzyme is denatured and the rate falls rapidly.
· Exam graph: rate rises gradually to an optimum, then drops steeply after denaturation.

The graph shows enzyme activity increasing with temperature until an optimum is reached. After the optimum, activity decreases sharply because the enzyme denatures and the active site is no longer complementary to the substrate. Source

pH and buffer solutions

· Each enzyme has an optimum pH where its active site shape is most suitable for substrate binding.
· Changes in pH alter hydrogen bonds and ionic bonds in the enzyme’s tertiary structure.
· This changes the shape of the active site, reducing enzyme–substrate complex formation.
· Extreme pH can cause denaturation.
· Use buffer solutions in pH investigations to keep pH constant.
· Exam graph: rate peaks at the optimum pH and decreases on either side.

The graph shows that enzyme activity is highest at an optimum pH. Activity decreases away from the optimum because changes in pH alter the bonding that maintains the enzyme’s active site shape. Source

Enzyme concentration

· If substrate concentration is in excess, increasing enzyme concentration increases rate.
· More enzyme molecules means more active sites available.
· More active sites allow more enzyme–substrate complexes to form per unit time.
· Rate is usually directly proportional to enzyme concentration while substrate is not limiting.
· If substrate becomes limiting, further increases in enzyme concentration have little or no effect.

Substrate concentration, Vmax and Km

· At low substrate concentration, increasing substrate increases rate because more active sites are occupied.
· At high substrate concentration, all active sites become occupied, so the enzyme is saturated.
· Once saturated, increasing substrate concentration does not increase rate further.
· The maximum possible rate is Vmax.
· Km is the substrate concentration at ½ Vmax.
· Km is used to compare enzyme affinity for substrates.
· A low Km means high affinity, because the enzyme reaches ½Vmax at a low substrate concentration.
· A high Km means low affinity, because more substrate is needed to reach ½Vmax.
· Exam graph: rate increases steeply at first, then levels off at Vmax.

This curve shows reaction rate increasing with substrate concentration before reaching a plateau. The plateau represents Vmax, when enzyme active sites are saturated with substrate. Source

Reversible inhibitors

· Reversible inhibitors bind temporarily to enzymes and reduce enzyme activity.
· The effect depends on inhibitor type and inhibitor concentration.
· Increasing inhibitor concentration usually decreases rate because fewer active enzyme molecules are available.
· In exam answers, always state whether the inhibitor is competitive or non-competitive and explain how this affects active site availability or enzyme shape.

Competitive inhibition

· A competitive inhibitor has a similar shape to the substrate.
· It binds to the enzyme’s active site.
· It prevents the substrate from binding, so fewer enzyme–substrate complexes form.
· Competitive inhibition can be reduced by increasing substrate concentration.
· At high substrate concentration, the substrate is more likely than the inhibitor to occupy active sites.
· Effect on kinetics: Vmax is unchanged, but Km increases.
· Reason: the same maximum rate can still be reached, but a higher substrate concentration is needed.

Non-competitive inhibition

· A non-competitive inhibitor binds away from the active site, at an allosteric site.
· This changes the enzyme’s tertiary structure and alters the shape of the active site.
· The substrate may no longer bind effectively, so fewer enzyme–substrate complexes form.
· Increasing substrate concentration does not overcome non-competitive inhibition.
· Effect on kinetics: Vmax decreases, but Km is unchanged for pure non-competitive inhibition.
· Reason: fewer active enzyme molecules are functional, but the affinity of remaining active sites is not changed.

The graph compares normal enzyme activity with competitive and non-competitive inhibition. Competitive inhibition increases Km but leaves Vmax unchanged, while non-competitive inhibition lowers Vmax. Source

Immobilised enzymes in alginate

· Immobilised enzymes are enzymes fixed or trapped in an inert material, such as alginate beads.
· In alginate immobilisation, enzyme solution is mixed with sodium alginate and dropped into calcium chloride solution.
· Calcium ions cause the alginate to form gel beads, trapping the enzyme inside.
· Compare immobilised and free enzymes by measuring reaction rate under the same conditions.
· Immobilised enzymes may show lower activity if substrate molecules diffuse slowly into the beads.
· Advantages: enzymes can be reused, products are easier to separate, enzyme contamination of the product is reduced, and enzymes may be more stable at different temperatures or pH values.
· Industrial advantage: immobilised enzymes can be used in continuous processes, where substrate flows over the enzyme and product is collected.

Investigating factors affecting enzyme activity

· Change only one independent variable: temperature, pH, enzyme concentration, substrate concentration or inhibitor concentration.
· Measure the dependent variable as reaction rate, such as volume of product per unit time or time for substrate disappearance.
· Keep control variables constant: enzyme volume, substrate volume, total volume, pH, temperature and reaction time.
· Use buffer solutions when investigating pH.
· Use a water bath to control temperature.
· Use repeats and calculate a mean rate.
· For reactions involving colour changes, use a colorimeter to measure absorbance or transmission more objectively.
· For rate calculations: rate = change ÷ time.
· When plotting graphs, use the independent variable on the x-axis and rate on the y-axis.