Enzyme Inhibitors, Cofactors and the Effects of pH, Temperature and Concentration

Enzymes are tightly regulated biological machines. Their activity can be modified by inhibitors, enhanced by cofactors, and affected by environmental conditions such as pH, temperature and substrate concentration. This lesson covers the OCR A-Level Biology A specification points 2.1.4 (c)–(g) — the effects of pH, temperature, enzyme and substrate concentration on the rate of enzyme-catalysed reactions; the roles of cofactors, coenzymes and prosthetic groups; and the effects of competitive and non-competitive inhibitors, including end-product inhibition.

This is the final lesson in the nucleic acids and enzymes topic. Together with Lesson 9, it completes the OCR specification content for Chapter 4 (in most OCR textbooks).

1. Factors Affecting Enzyme Activity

Enzyme activity (measured as the rate of reaction, usually the rate at which substrate is used up or product is formed) is affected by several factors:

Temperature
pH
Enzyme concentration
Substrate concentration
Presence of inhibitors
Presence of cofactors, coenzymes or prosthetic groups

OCR expects you to be able to describe, explain and draw graphs of each of these effects.

2. Effect of Temperature

 Rate ^
      |          *
      |        *   \
      |      *      \
      |    *         \
      |  *            \
      |*_______________\__________
      |                 denatured
      +--------------->
                   Temperature
              (optimum around 37-40°C for
               most mammalian enzymes)

As temperature increases, molecules gain kinetic energy. Enzyme and substrate molecules collide more frequently and with more energy, so the number of successful enzyme–substrate complexes formed per second increases. The rate rises.
Each enzyme has an optimum temperature (around 37 °C for most human enzymes, much higher for enzymes in thermophilic bacteria).
Above the optimum, the increased vibration of atoms in the enzyme begins to break the weak bonds (hydrogen bonds and ionic bonds) that hold the tertiary structure together. The active site changes shape, and eventually the enzyme denatures and loses all activity.
Denaturation is usually permanent — cooling a denatured enzyme does not restore its function.

Q10

The temperature coefficient (Q10) is the factor by which a reaction rate increases for a 10 °C rise in temperature. For most enzymes below the optimum, Q10 ≈ 2 (the rate roughly doubles for every 10 °C rise).

3. Effect of pH

 Rate ^
      |          *
      |        *   *
      |       *     *
      |      *       *
      |     *         *
      |    *           *
      |_*_______________*______
      +--------------->
                   pH
         (optimum varies by enzyme:
          pepsin ≈ 2, trypsin ≈ 8)

Each enzyme has an optimum pH, at which the active site has its ideal 3D shape and charge distribution.
Either side of the optimum, the rate decreases.
Why? pH is a measure of H⁺ concentration. Changes in pH alter the charges on acidic (–COOH) and basic (–NH₂) side chains in the enzyme, disrupting the hydrogen bonds and ionic bonds that maintain the tertiary structure. The active site changes shape; the substrate can no longer bind properly.
Extreme changes cause denaturation, again usually permanent.

Examples of Optimum pH

Enzyme	Location	Optimum pH
Pepsin	Stomach	~2
Salivary amylase	Mouth	~7
Trypsin	Small intestine	~8

Exam Tip: Always refer to specific bonds (hydrogen and ionic bonds) in the tertiary structure when explaining pH effects. Just saying "the enzyme stops working" does not score marks.

4. Effect of Enzyme Concentration

 Rate ^
      |              __________
      |            /
      |           /
      |          /     (substrate limiting above this point)
      |         /
      |        /
      |       /
      |______/________________
      +--------------->
                   [Enzyme]

Provided substrate is in excess, increasing enzyme concentration increases the rate of reaction in direct proportion — more active sites are available, so more ES complexes form per second.
Eventually the rate levels off because the substrate concentration becomes limiting — every substrate molecule can find an active site, and adding more enzyme makes no difference.

5. Effect of Substrate Concentration

 Rate ^
      |              __________
      |            /  (Vmax)
      |           /
      |          /
      |         /
      |        /   (all active sites occupied)
      |       /
      |______/________________
      +--------------->
                 [Substrate]

At low substrate concentrations, most active sites are empty — adding more substrate gives a proportional increase in rate.
As substrate concentration increases, the rate levels off because all the active sites are occupied at any given moment. The enzyme is working at maximum rate (Vmax).
Adding more substrate beyond this point has no further effect — only adding more enzyme can increase the rate.

6. Cofactors, Coenzymes and Prosthetic Groups

Some enzymes are fully active on their own. Many others need helper molecules to work properly. These helpers are called cofactors (a general term) and come in several forms.

Key Definition — Cofactor: A non-protein substance that is needed for the proper functioning of an enzyme. Cofactors may be inorganic ions, organic coenzymes or prosthetic groups.

6.1 Inorganic Ion Cofactors

These are simple metal ions that bind to the enzyme or substrate to enable catalysis.

Cl⁻ is a cofactor for amylase.
Mg²⁺ is a cofactor for many ATP-using enzymes, including DNA polymerase and kinases.

6.2 Coenzymes

Coenzymes are organic molecules that bind temporarily to an enzyme (or, more often, shuttle between enzymes) to transfer a chemical group. They are not permanently attached.

Most coenzymes are derived from vitamins — which is why vitamin deficiencies cause metabolic problems.

Coenzyme	Role	Vitamin origin
NAD (nicotinamide adenine dinucleotide)	Carries hydrogen (reducing power) in respiration	Niacin (B3)
FAD (flavin adenine dinucleotide)	Carries hydrogen in respiration	Riboflavin (B2)
Coenzyme A (CoA)	Carries acetyl groups in respiration	Pantothenic acid (B5)

Exam Tip: NAD, FAD and Coenzyme A are all coenzymes that you need to know for OCR — they will reappear in the Year 2 topic on respiration.

6.3 Prosthetic Groups

Prosthetic groups are non-protein components that are permanently, covalently bound to an enzyme and are essential for its function.

Prosthetic group	Enzyme	Role
Fe²⁺ (haem)	Catalase	Needed to break down hydrogen peroxide
Zn²⁺	Carbonic anhydrase	Needed to interconvert CO₂ and HCO₃⁻ in red blood cells

Lesson 10: Enzyme Inhibitors, Cofactors and the Effects of pH, Temperature and Concentration