Enzyme action and investigating pH

The active site has a shape complementary to its substrate. Complementary means that the shapes fit together, rather than being identical. This explains enzyme specificity: a substrate with an unsuitable shape cannot fit the active site, so the enzyme cannot catalyse its reaction.

The lock and key model represents this relationship. The enzyme is like a lock and its substrate is like the matching key. A particular key fits a particular lock, just as an appropriate substrate fits an enzyme's active site. This is a simplified model of molecular interactions, not a suggestion that enzymes are rigid metal objects.

From substrate to products

Enzymes and substrates move randomly in solution. When a suitable substrate collides with the enzyme's active site and binds, an enzyme–substrate complex forms. The enzyme catalyses the reaction, converting the substrate into a product or products.

The products then leave the active site. The enzyme is unchanged and its active site is available for another substrate. A breakdown reaction can therefore be represented as a repeating sequence: substrate binds, products form, products leave, and the enzyme is reused.

Temperature changes both movement and shape

At low temperatures, enzyme and substrate molecules have less kinetic energy and move more slowly. There are fewer successful collisions with active sites each second, so the reaction proceeds slowly.

As temperature rises towards the enzyme's optimum temperature, molecules gain kinetic energy and move faster. More successful collisions occur each second, increasing the reaction rate. The optimum is the temperature at which the reaction is fastest under the conditions being investigated. Many human enzymes work best at about 37°C; this is not a universal optimum for every enzyme.

At sufficiently high temperatures, bonds maintaining the enzyme's three-dimensional shape are disrupted. The active site changes shape and is no longer complementary to the substrate. Fewer enzyme–substrate complexes can form, so the reaction rate falls. This change is called denaturation.

Cooling and overheating therefore slow enzyme activity for different reasons. Cooling reduces molecular movement; overheating can change the active site's shape.

Each enzyme has an optimum pH

The pH scale describes how acidic or alkaline a solution is: below pH 7 is acidic, pH 7 is neutral and above pH 7 is alkaline. An enzyme's optimum pH is the pH at which it catalyses its reaction fastest under the conditions being investigated.

Different enzymes have different optimum pH values. For example, some stomach enzymes work best in acidic conditions around pH 2, whereas some enzymes in the small intestine work best in alkaline conditions around pH 8–9. Neutral conditions are therefore not best for every enzyme.

Moving away from an enzyme's optimum pH reduces its activity. Changes in pH can disrupt the bonds maintaining the protein's shape, altering the active site so that the substrate binds less effectively. At sufficiently extreme pH values, the enzyme becomes denatured and can no longer catalyse the reaction.

On a graph of reaction rate against pH, the peak identifies the optimum. The rate falls on either side as conditions become less suitable for that enzyme.

Investigating pH using continuous sampling

Required practical 4 investigates how pH affects the rate at which amylase, an enzyme that breaks down starch into sugars, digests a starch solution.

Iodine reagent provides a way to follow the disappearance of starch. It turns blue-black when starch is present and remains orange-brown when no starch is detected. Testing repeated samples lets you determine the time taken for complete starch digestion.

The apparatus includes test tubes and a rack, a spotting tile, pipettes for sampling, syringes or measuring cylinders for measuring volumes, a timer, a thermometer and a water bath or electric heater. The solutions needed are amylase, starch, iodine and buffers of different pH values. A buffer solution maintains the chosen pH during the reaction.

1. Place separate drops of iodine in the wells of a spotting tile. Label the reaction tube with the pH being tested.
2. Measure the volumes of amylase, starch and buffer. Bring the solutions to the same controlled temperature using the water bath or electric heater, checking the temperature with a thermometer.
3. Mix the amylase and buffer, then add the starch. Mix and start the timer immediately: the reaction begins when enzyme and substrate meet. Keep the reaction tube at the controlled temperature.
4. Every 30 seconds, remove a small sample and add it to a fresh iodine drop. Use clean sampling equipment to avoid contamination.
5. Record the time of the first sample in which iodine stays orange-brown. This is the endpoint: starch is no longer detected.
6. Repeat at a range of pH values, and repeat trials at each pH to calculate a mean digestion time and check for anomalous results.

This repeated removal and testing of samples is called continuous sampling. Sampling every 30 seconds limits the precision of the endpoint: starch may have disappeared between the last blue-black sample and the first orange-brown sample.

The pH is the independent variable; digestion time is the measured dependent variable. Keep the concentrations and volumes of starch and amylase, buffer volume and temperature constant. Temperature must be controlled using a water bath or electric heater because temperature itself affects enzyme activity. Changing both temperature and pH would prevent you from confidently attributing a difference in time to pH alone.

Calculating and comparing reaction rates

A reaction rate describes how much chemical change occurs per unit time. An average rate can be calculated from either reactant disappearance or product formation:

$$\text{mean rate} = \frac{\text{amount of reactant used up}}{\text{time taken}}$$

or

$$\text{mean rate} = \frac{\text{amount of product formed}}{\text{time taken}}$$

These are alternative measurements of rate; their numerical values are not necessarily equal. Choose the expression that matches the quantity measured.

The units depend on the measurement. Mass in grams divided by time in seconds gives g/s; a volume in cm³ divided by seconds gives cm³/s.

For an enzyme reaction, suppose 0.60 g of product forms in 120 s. Its mean rate is $0.60 \div 120 = 0.0050\text{ g/s}$. This means that, on average, 0.0050 g of product formed each second over that interval.

In the amylase investigation, iodine indicates an endpoint rather than measuring the mass of starch digested. When the same initial amount of starch is used in every trial, compare relative rates using reciprocal digestion time:

$$\text{relative rate} = \frac{1}{\text{digestion time}}$$

For example, digestion times of 60 s and 120 s give relative rates of $1/60 = 0.0167\text{ s}^{-1}$ and $1/120 = 0.00833\text{ s}^{-1}$. The 60 s reaction is twice as fast because it reaches the same endpoint in half the time.

Plot relative rate against pH to compare activity. The pH giving the highest rate is the fastest of those tested; testing more closely spaced pH values around that peak would locate the optimum more precisely.

• Below optimum temperature: warming increases kinetic energy and successful collisions, increasing rate.
• High temperature: denaturation alters the active site; the substrate no longer fits effectively.
• Low temperature slows activity without denaturing the enzyme.
• Optimum pH varies between enzymes. Activity decreases on either side; sufficiently extreme pH can cause denaturation.

Required practical 4

• Change pH using buffers; measure the time for amylase to digest starch completely.
• Control temperature with a water bath or electric heater; keep solution volumes and enzyme/starch concentrations constant.
• Start timing when starch and amylase mix. Sample into fresh iodine drops every 30 seconds.
• Blue-black = starch present. First orange-brown sample = endpoint.
• Repeat each pH and calculate mean digestion times. Sampling interval limits endpoint precision.

Rate calculations

$$\text{mean rate} = \frac{\text{reactant used up or product formed}}{\text{time taken}}$$