Cambridge Syllabus Notes

5.1 Enzymes – How pH Affects Enzyme Activity

Learning objectives (AO1‑AO3)

Define a catalyst and an enzyme, and explain why enzymes are essential for life.
Describe the structure of an enzyme (protein) and the role of the active site.
State the optimal pH of an enzyme and explain how pH influences activity (fit vs denaturation).
Analyse how changes in pH alter the ionisation of amino‑acid side‑chains and consequently the three‑dimensional shape of the enzyme.
Link the theory to the required practical investigation of pH effects on enzyme activity.
Apply the knowledge to typical IGCSE exam questions (AO2/AO3).

1. What is a catalyst? What is an enzyme?

Catalyst: a substance that speeds up a chemical reaction without being consumed in the overall reaction.
Enzyme: a biological catalyst – a specialised protein that lowers the activation energy of a specific biochemical reaction.
Enzymes are not permanently altered; they can be used repeatedly as long as their three‑dimensional structure remains intact.

2. Why are enzymes vital for life?

Most metabolic reactions are thermodynamically favourable but are kinetically slow; enzymes increase the rate so that reactions occur at a speed compatible with life.
Examples: digestion of food, synthesis of DNA, cellular respiration, muscle contraction.

3. Enzyme structure and the active site

Enzymes are proteins folded into a specific three‑dimensional shape (primary → tertiary structure).
The active site is a small region where the substrate binds.
Fit between enzyme and substrate is often described by the lock‑and‑key model, but many enzymes show induced fit – the active site adjusts slightly when the substrate approaches.

4. How pH influences enzyme activity

4.1 Role of pH

pH determines the ionisation state of ionisable side chains such as –COOH (acidic), –NH₂ (basic) and –SH (thiol).
Changing the charge on these groups alters hydrogen‑bonding, ionic interactions and, at very extreme pH, can affect disulphide bridges (normally stable in the physiological pH range).
These changes modify the overall shape of the protein and, most importantly, the geometry of the active site.

4.2 Optimal pH – “fit”

Each enzyme has a pH at which its active site has the correct shape for maximum substrate binding – the optimal pH.
At the optimal pH the enzyme works at its highest rate (V_max).
Moving away from this pH reduces the rate because the fit becomes poorer (lower affinity, fewer effective collisions). This effect is usually reversible if the pH returns to the optimum.

4.3 Denaturation by pH

Very acidic (pH ≈ 0‑2) or very alkaline (pH ≈ 12‑14) conditions disrupt the non‑covalent forces that maintain the protein’s tertiary structure.
When the three‑dimensional shape is lost, the active site is destroyed – the enzyme is **denatured** and activity falls essentially to zero.
Denaturation is generally irreversible under physiological conditions; the enzyme cannot be reused.
If the pH change is only moderate and short‑lived, activity can be restored when the pH returns to the optimum – this is a reversible effect, not denaturation.

4.4 Example enzymes required by the IGCSE syllabus

The three enzymes that must be known, together with their optimal pH values, are:

Pepsin – works best at pH 2 (highly acidic environment of the stomach).
Trypsin – works best at pH 8 (alkaline environment of the small intestine).
Salivary amylase – works best at pH 7 (neutral environment of the mouth).

5. Practical investigation – “Effect of pH on enzyme activity”

This practical satisfies the syllabus requirement for a skills investigation.

Step	What to do	Key points / safety
1. Prepare buffers	Make a series of pH 4, 5, 6, 7, 8, 9, 10 buffers (e.g., acetate, phosphate, tris).	Label clearly; wear gloves and goggles.
2. Prepare enzyme solution	For pepsin – dissolve a measured amount of powdered pepsin in distilled water; keep on ice.	Pepsin is acidic – handle with care.
3. Add substrate	Add a fixed volume of substrate (e.g., haemoglobin for pepsin) to each test tube.	Keep substrate concentration the same in all tubes.
4. Start the reaction	Add a fixed volume of the appropriate buffer to each tube, then quickly add the enzyme solution to start the reaction.	Start timing immediately; record the time.
5. Measure activity	Measure the rate of product formation (e.g., increase in absorbance at 280 nm) or the time taken for a colour change.	Take the same number of readings for each pH.
6. Record & plot	Calculate relative activity (percentage of the highest rate) and plot activity vs pH.	Result should be a bell‑shaped curve.

6. Graphical representation – bell‑shaped activity‑pH curve

Students should be able to reproduce the following sketch in an exam.

7. Summary of key points

Enzymes are protein catalysts that speed up reactions without being consumed.
Each enzyme has a characteristic optimal pH at which the active site fits the substrate best, giving maximum activity.
pH changes the ionisation of side‑chains, altering hydrogen‑bonding and ionic interactions, which can modify the enzyme’s shape.
Small deviations from the optimum cause a reversible reduction in activity (the “fit” is poorer).
Very high or very low pH disrupts non‑covalent forces, leading to irreversible denaturation and loss of activity.
Typical IGCSE examples: pepsin (pH 2), trypsin (pH 8), salivary amylase (pH 7).

8. Common misconceptions (teacher notes)

“Enzymes are destroyed by any change in pH.” – Only extreme pH values cause irreversible denaturation; moderate changes merely lower the rate.
“All enzymes work best at pH 7.” – Only enzymes that function in neutral cellular compartments have this optimum; digestive enzymes have acidic or alkaline optima.
“Denaturation means the enzyme is chemically broken down.” – Denaturation is loss of three‑dimensional shape, not cleavage of peptide bonds (unless very harsh conditions are used).
“Reversible inhibition” vs “reversible effect”. – In the IGCSE syllabus the term used is *reversible effect* (activity falls when pH moves away from optimum but can be restored). The word “inhibition” is not required here.

9. Exam technique – answering AO2/AO3 questions

State the fact (AO2). Example: “Pepsin has an optimal pH of about 2.”
Explain the reason (AO3). Discuss ionisation of side‑chains, effect on hydrogen‑bonding/ionic interactions, resulting change in active‑site geometry, and why activity falls or why denaturation occurs at extreme pH.
Use precise terminology: optimal pH, active site, substrate, fit, denaturation, reversible effect.
If a diagram is required, sketch a labelled bell‑shaped curve (see Section 6).

10. Sample IGCSE exam question and mark scheme

Question: a) State the effect of moving a solution from pH 7 to pH 3 on the activity of pepsin. b) Explain why the activity changes, referring to the structure of the enzyme.

Suggested answer (6 marks)

AO2 – 2 marks – “The activity of pepsin increases because pH 3 is closer to its optimal pH (≈ 2).”
AO3 – 4 marks – “At pH 3 the ionisable side‑chains of pepsin (e.g., carboxyl groups) are predominantly in the protonated form, allowing the correct pattern of hydrogen‑bonding and ionic interactions that maintain the enzyme’s tertiary structure. The active site therefore has the proper geometry to bind substrate efficiently, giving a higher rate. If the pH were lowered further (e.g., pH 1) the extreme acidity would disrupt these non‑covalent forces, causing denaturation and a loss of activity.”

11. Quick revision checklist

Definition of catalyst and enzyme (protein).
Why enzymes are needed for life.
Active site – lock‑and‑key vs induced fit.
Ionisable side‑chains (‑COOH, ‑NH₂, ‑SH) and how pH changes their charge.
Optimal pH = maximum fit → maximum activity.
Moving away from optimum = reversible reduction in activity.
Extreme pH = irreversible denaturation (loss of non‑covalent forces).
Key examples and their optimal pH: pepsin ≈ 2, trypsin ≈ 8, salivary amylase ≈ 7.
Sketch a labelled bell‑shaped activity‑pH curve.
Recall the practical steps for investigating pH effects on enzyme activity.

Explain pH effects on enzyme activity: fit and denaturation.