Factors Affecting Enzyme Activity
Enzymes are complex proteins and their structure can be affected by factors such as temperature and pH - causing changes in the shape of their active site.
Factors affecting enzyme activity can be investigated by measuring rates of reaction they catalyse.
Temperature
In an enzyme‑controlled reaction, increasing temperature results in more frequent collisions between the substrate and the active site, increasing the rate of reaction.
Temperature coefficient (Q10) of a reaction = a measure of how much rate of reaction increases with a 10 °C rise in temperature. For enzyme controlled reactions, it is usually 2 (meaning rate of reaction doubles with 10 °C temp increase).
Denaturation from temperature
Structure can be affected by temperature. At higher temperatures, bonds holding proteins together vibrate more until eventually breaking the bonds ( if temp = too high ) causing in a ‘Denatured’ tertiary structure of the enzyme.
This means active site changes shape and no longer complementary to the substrate and therefore no longer a catalyst.
Optimum Temperature
Temperature at which enzyme has highest rate of activity before denaturing. Can vary significantly dependant on the enzyme.
Many enzymes in human body = 40°C
Thermophilic bacteria (in hot springs) have enzymes with optimum temperatures of 70°C
psychrophilic organisms (live in areas of cold) have optimum temperatures below 5°C.
Once enzymes have denatured above optimum temperature, the decrease in rate of reaction is rapid as only a slight change in shape of active site to not be able to be complementary to its substrate (and Temperature coefficient is irrelevant as enzymes have denatured).
decrease in rate of reaction below optimum temperature are less rapid. As enzymes haven’t denatured, only less active.
Temperature Extremes
Living organisms evolve to cope with living their environments (within certain temperature range)
Example → Extremely cold environments like deep oceans, high altitudes, polar regions. Enzymes adapted to the cold have more flexible structures, especially the active site, making them less stable than enzymes working at higher temperatures. Smaller temperature changes will denature them.
Example → Thermophiles = organisms adapted to living in very hot environments like hot springs. These enzymes are more stable do to increased number of bonds. The shapes of the enzymes , particularly the active sites, are more resistant to change as temperature rises.
Siamese Cats → Provide visual evidence of the effect of temperatures on enzyme activity. An enzyme known as ‘Tyrosinase’, responsible for catalysing production of melanin. Due to mutation, Siamese cats produce a mutated form of this enzyme that is denatured and therefore inactive at normal body temperature (too high) meaning their fur is white or cream coloured. Some features of these cats (tail, ears, nose etc) are a lower temperature - too low to denature mutant tyrosinase. This leads to darker features on these parts of the body |
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pH
Proteins (so Enzymes) - affected by changes in pH . hydrogen bonds and ionic bonds between amino acid R groups hold proteins in their 3D shape. These bond result from interactions between polar and charged R groups present on the amino acids, forming the tertiary structure
A change in pH = change in hydrogen ion concentration. More Hydrogen ions present in low pH (acidic) vice versa.
Active site will only be in right shape at certain hydrogen ion concentration = optimum pH .
When pH changes from optimum, the active site is altered. However, if pH returns to optimum again, active site returns to normal shape and catalyse a reaction again . This is known as ‘Renaturation’
When pH changes significantly, the structure of the enzyme is irreversibly altered and the active site is no longer complementary to its substrate. The enzyme is denatured, reducing the rate of reaction.
When the pH changes, the hydrogen ions interact with polar and charged R groups. Therefore, changing the concentration of hydrogen ions changes the degree of this reaction. This can also affect the interactions of R groups with each other. Shown Below
The more hydrogen ions present (low pH), the more hydrogen ions attach to polar and charged R‑groups. This prevents R‑groups from interacting with each other, so hydrogen and ionic bonds break and the enzyme’s tertiary structure changes.
At high pH, there are too few hydrogen ions to maintain the charges on R‑groups, For example, positively charged R‑groups can lose H⁺ and become neutral, and negatively charged R‑groups remain strongly negative. Because their charges change, these R‑groups can no longer form ionic bonds with each other. This causes ionic interactions to weaken or break, altering the enzyme’s tertiary structure.
Because both extremes disrupt R‑group interactions, enzymes only function within a narrow pH range.
Substrate and Enzyme concentration
Concentration of Substrate increasing = higher collision rate with active sites of enzymes and more enzyme - substrate complexes. Rate of reaction increases
Concentration of enzyme increases = increase number of available active sites in particular volume meaning more enzyme-substrate complexes at a faster rate.
Rate of reaction increases up to its max (Vmax). At this point, all active sites are occupied by substrate particles and no more enzyme substrate complexes can be formed until products are released from active sites
The only way to increase Vmax would be to add more enzyme or increase temperature
Serial Dilutions
A Serial Dilution is a repeated, stepwise dilution of a stock solution of known concentration .
To produce a range of concentrations usually in factors of 10 (dilution factor of 10)
Used to investigate the effect of changing the concentration of an enzyme or substrate in a reaction.
Image here
Adding 1ml of stock solution to 9ml of distilled water gives 10ml of dilute solution in which there is 1/10ml stock (hence a 10 fold dilution or 1/10).
This whole step is repeated a number of times to give a serial solution.
Catalase is an enzyme that catalyses the breakdown of Hydrogen peroxide. To investigate effect of different concentrations of catalase on the rate of breakdown of hydrogen peroxide, catalase-rich tissues such as liver can be ground down to make a solution containing catalase from the cells .
Serial dilutions of this solution will contain different concentrations of catalase. The effect of the different concentrations on rate of reaction can be investigated by adding equal volumes of hydrogen peroxide to each solution.
New Concentration = Old Concentration/10