Factors Affecting Enzyme Activity: Temp, pH, and Substrate
Enzyme activity depends on temperature, pH, substrate concentration and enzyme concentration: the rate rises with each factor up to a limit, and extreme temperature or pH denatures the enzyme and stops it from working. In brief: most human enzymes work best near 37 °C and pH 7, but there are exceptions: pepsin works best at about pH 2. Raising the temperature increases the rate until the optimum, then the rate falls sharply as the enzyme is denatured. At high substrate concentration, the rate levels off because all active sites are in use.
Understanding these factors is essential for medicine, food processing and laboratory work. This guide explains the effect of each factor, including the shapes of the graphs, defines denaturation, compares the factors in a table, works through a numerical experiment on substrate concentration and temperature coefficient (Q₁₀) and corrects common mistakes.
How does temperature affect enzyme activity?
At low temperatures, molecules have little kinetic energy, so they move slowly, collide rarely and few collisions have enough energy to react. As temperature rises, collisions between enzyme and substrate become more frequent and more energetic, so the rate increases. For many reactions, a 10 °C increase roughly doubles the rate.
Above the optimum temperature, the rate falls because the enzyme begins to denature. The optimum for most human enzymes is about 37 °C. Some enzymes work at higher temperatures: the DNA polymerase of Thermus aquaticus, a bacterium from hot springs, works at about 72 °C and is used in PCR. Enzymes of Arctic fish work well near 0 °C. The graph of rate against temperature is a skewed curve that rises slowly, peaks and drops steeply.
How does pH affect enzyme activity?
The charges on the amino acid side chains of an enzyme depend on pH, so changes in hydrogen ion concentration disrupt ionic and hydrogen bonds that maintain the shape of the active site. Each enzyme has an optimum pH, at which its activity is the highest. The graph is a bell-shaped curve.
- Pepsin (stomach): pH about 1.5 to 2.
- Salivary amylase: about pH 6.8 to 7.
- Trypsin and pancreatic enzymes (small intestine): about pH 8.
- Most intracellular enzymes: close to pH 7.
A small change away from the optimum slows the enzyme (and the change is reversible), but a large change causes irreversible denaturation. This is the reason that salivary amylase stops working in the acid of the stomach.
How does substrate concentration affect enzyme activity?
At a low substrate concentration, there are many free active sites, and each collision adds a new enzyme-substrate complex. The rate is nearly proportional to the substrate concentration. As the concentration rises, more active sites are occupied. Eventually, all the active sites are busy at any time (the enzyme is saturated), and adding more substrate does not increase the rate. The rate reaches a maximum, Vmax, which is limited by the amount of enzyme and the speed with which it turns over the substrate. The graph is a curve that rises steeply and then flattens (a hyperbola). The substrate concentration at which the rate is half of Vmax is the Michaelis constant, Km, which is lower for enzymes with a higher affinity for the substrate.
How does enzyme concentration affect the rate?
If substrate is in excess, the rate is directly proportional to the enzyme concentration, because more enzyme molecules mean more active sites. Doubling the amount of enzyme doubles the rate. If the substrate becomes limiting, the rate stops increasing and levels off.
What is denaturation?
Denaturation is the loss of the three-dimensional structure of a protein, as a result of the breaking of the weak bonds (hydrogen bonds, ionic bonds and hydrophobic interactions) that hold the folded shape. The peptide bonds of the primary structure are not broken. The shape of the active site changes, so the substrate no longer fits and the enzyme loses its activity. Heat, extreme pH and some chemicals, such as heavy metals and alcohol, denature proteins. The effect is usually irreversible, as when an egg white solidifies on cooking. A few proteins can refold when conditions return to normal.
Effects of the four factors compared
| Factor | Effect when increased from low | Shape of graph | What limits the rate at the plateau or fall |
|---|---|---|---|
| Temperature | Rate rises, then falls | Peak, then steep drop | Denaturation above optimum |
| pH | Rate rises to the optimum, then falls | Bell-shaped curve | Change in charges and shape |
| Substrate concentration | Rate rises, then levels off | Hyperbola | All active sites in use |
| Enzyme concentration | Rate rises in proportion | Straight line, then plateau | Substrate becomes limiting |
Worked example: rates and Q₁₀
Substrate concentration
An enzyme experiment gives the following initial rates of product formation.
| Substrate (mmol/L) | Rate (µmol/min) | Increase from previous row |
|---|---|---|
| 1 | 20 | – |
| 2 | 36 | 16 |
| 4 | 58 | 22 |
| 8 | 75 | 17 |
| 16 | 83 | 8 |
| 32 | 86 | 3 |
- The rate rises steeply at low concentrations and then levels off near 86 to 90 µmol/min, which suggests Vmax is about 90 µmol/min.
- At low concentration (1 to 2 mmol/L), doubling the substrate increased the rate from 20 to 36, which is 1.8 times, close to proportional.
- At high concentration (16 to 32 mmol/L), doubling the substrate only increased the rate from 83 to 86, which is 1.04 times. The enzyme is saturated.
- Km is the concentration at which the rate is half of Vmax (about 45 µmol/min). By interpolation between 2 mmol/L (36) and 4 mmol/L (58), the rate of 45 is reached at about 2 + 2 × (45 − 36) ÷ (58 − 36) = 2 + 0.82 ≈ 2.8 mmol/L.
Temperature coefficient
The Q₁₀ is the factor by which the rate increases for a rise of 10 °C: Q₁₀ = rate at (T + 10) ÷ rate at T. If an enzyme reaction has a rate of 12 units at 20 °C and 26 units at 30 °C, then Q₁₀ = 26 ÷ 12 ≈ 2.2. A value of about 2 is typical for enzyme reactions. Q₁₀ applies only below the optimum temperature, because above the optimum, denaturation reduces the rate.
How can you investigate enzyme activity?
A good experiment changes one factor and keeps all the others constant. For the effect of pH on amylase, the investigator mixes starch with amylase and buffer solutions of different pH in a water bath at 37 °C, and takes samples every 30 seconds into iodine solution. The time taken for the blue-black color to disappear is measured. The rate is 1 ÷ time. Buffers keep the pH constant. Repeating each condition at least three times and calculating the mean gives more reliable results. Safety requires care with hot water and chemicals.
How were these relationships discovered?
Leonor Michaelis and Maud Menten published their equation for the effect of substrate concentration in 1913, and S. P. L. Sørensen introduced the pH scale in 1909 while working on enzymes at the Carlsberg brewery in Copenhagen. Jacobus van ‘t Hoff and Svante Arrhenius studied the effect of temperature on reaction rates in the 1880s. Peptic and tryptic enzymes were characterized by John Northrop in the 1930s, for which he shared the 1946 Nobel Prize in Chemistry.
Which mistakes are common?
Mistake 1: Saying that low temperature denatures enzymes
Cold slows the reaction but does not usually denature the enzyme. The effect is reversible.
Mistake 2: Explaining the plateau by saying that “the enzyme gets tired”
The plateau occurs because all active sites are occupied (saturation).
Mistake 3: Assuming that all enzymes have the same optimum pH
The optimum depends on the environment where the enzyme works.
Mistake 4: Not controlling the other variables
If temperature, pH and concentrations are not controlled, the effect of the factor being studied cannot be identified.