How Does pH Affect Enzyme Activity? (Graph & Experiment)

Effect of pH on enzyme activity

This lecture and experiment will look at pH’s effects on enzyme activity. In chemistry, pH calculates the relative proportions of hydrogen plus (H+) and hydroxide minus ions (OH−).

Simply pH is a substance’s acidity or acidic when a solution is acidic, such as your stomach’s contents. It means many hydrogen ions and atoms floating around in your stomach have lost their electrons and become positive. If you compare that solution to a glass of water, the water contains far fewer hydrogen protons, mostly water molecules.

Enzymes speed up chemical reactions. They have a groove on their surface called the active site to do this. The enzyme breaks down the substrate into products. The substrate must fit perfectly into the lock and key theory active site. Enzymes are very fussy molecules with the right pH to work in a good environment. We will describe the effect of pH and enzyme activity by looking at the graph.

What is pH?

The pH of a solution is the measure of hydrogen ion concentration. Each enzyme has an optimum pH. Many ionic bonds hold the tertiary structure of an enzyme and, therefore, the active site’s shape.

Each enzyme has a range of pH values called its optimum pH, at which the reaction rate is catalyzed fastest. The reason pH has such an important effect on enzymes is their proteins. The change in pH will change the bonding patterns; as a result, increased changes from the optimum pH will change the shape of the active site.

When the temperature rises too high in enzyme-controlled reactions, the change in the active site is irreversible. It is permanent. So it causes a far more drastic change in the rate of reaction.

How Does pH Affect Enzyme Activity?

pH can significantly affect enzyme activity. Enzymes are biological catalysts that facilitate chemical reactions in living organisms. They have an optimum pH range, where their activity is maximized. Deviations from this optimal pH can impact enzyme activity in the following ways:

Effect on Enzyme Structure: Enzymes have a specific three-dimensional structure crucial for their activity. pH can alter an enzyme’s ionic and hydrogen bonding interactions, causing changes in its overall structure. If the pH deviates significantly from the optimum range, the enzyme’s structure can be disrupted, leading to denaturation or loss of its catalytic activity.

Ionization of Substrate and Active Site: The pH affects the ionization states of both the enzyme and its substrate. The active site of an enzyme often contains amino acid residues with ionizable groups that participate in catalysis. When the pH is not optimal, the ionization of these groups can be disrupted, affecting the binding of the substrate to the active site and the subsequent catalytic reactions.

Optimal pH Range: Each enzyme has an optimal pH range exhibiting maximum activity. This pH range depends on the specific enzyme and the environment in which it functions. Deviating from this optimal pH can decrease enzyme activity. For example, an enzyme with an optimal pH of 7 (neutral pH) may have reduced activity in a more acidic (lower pH) or alkaline (higher pH) environment.

Influence on Charge Distribution: pH affects the distribution of charges on the enzyme’s active site and the substrate. The active site’s charge distribution is crucial for substrate binding and catalysis. Deviations from the optimal pH can disrupt the charge interactions between the enzyme and substrate, impacting the enzyme’s ability to bind the substrate and perform catalytic reactions.

The pH measures the acidity or basicity of a substance or a solution. Our stomach produces powerful acids that acid helps us start to digest our foods. This acid helps us digest but also kills harmful microorganisms we eat from our food.

Our blood has a pH of 7.4, so that’s quite neutral and very similar to water. One of the few basic foods we eat is baked beans; we all know you may produce a little more gas.

That’s because of basic food’s effect on our digestive system. It doesn’t mean that it’s bad for us. It’s still healthy to eat acids. What are acids? They are substances with hydrogen ions and have a pH lower than 7.

pH Scale:

  • pH 7 – Neutral.
  • pH 0 to 6 – Acidic
  • pH 8 to 14 – Alkaline

If you go above or below the pH optimum for each enzyme, you can see that the reaction rate decreases. This is because the enzyme denatures. It changes shape above or below the optimum pH.

  • If you have below 9 pH, the enzyme’s active site denatures. So above pH 7 active site is denaturing.
  • The active site will denature if you have below pH 7 in more acidic conditions. Therefore, the substrate can no longer bind, the product form decreases, and the reaction rate decreases.

Results: The enzyme’s active site is natural if you increase or decrease the pH above or below the optimum. The active site changes shape, and the substrate no longer binds. Therefore, less product is formed, and the reaction rate will decrease. The rate of reaction will decrease.

Enzymes work best with a narrow pH range. Any variation above or below a specific level reduces their rate of activity.

Examples of enzymes: Pepsin, Trypsin, Amylase, Rennin, etc.

pH for Pepsin: Pepsin is a powerful enzyme that digests proteins in the stomach. It digests meat, eggs, seeds, or dairy products and breaks them into peptides. These peptides are absorbed from the intestine to the bloodstream or broken down further by pancreatic enzymes such as trypsin.

Its optimal pH is 2. Its pH is around 2 because the stomach’s pH is about 1.5 to 3.

pH for Trypsin: Trypsin works at six to seven alkalinity levels or acidity outside the optimum pH—enzymes in the digestive tract function in an acidic or alkaline environment.

pH for Amylase: It breaks down starch and best works in a weak acid condition. So its pH is around 6.7, but it can also range up to pH 7, which is neutral.

The action of amylase on starch stops when the food passes into the stomach. It is because the low pH of the gastric juice makes it inactive. So the amylase cannot work or perform its function with a pH of 1.5 to 3.

pH for Rennin: The digestive protein enzyme rennin is found in gastric juice in the stomach function. It’s turning milk, a liquid, into a solid. The purpose of this is so that you’re able to absorb all the nutrients in that solid.

If it were to stain as a liquid, it would pass through the body, and you wouldn’t be able to absorb the proteins and nutrients from the milk. A commercial form of renin is called rennet.

Rennin has an optimum pH of 3.4. At values pH above 7, it loses activity rapidly.

The graph of pH affects enzyme activity.

Here is a graph of increasing enzyme activity on the Y-axis against increasing pH on the X-axis. The shape of the curve denotes the enzyme activity increases. It reaches a maximum and then decreases. What’s happening at points X, Y, and Z?

pH affect on enzyme activity graph
pH effect on enzyme activity graph
  • At point X, which is low pH. The enzyme is protonated, which means it has a positive charge. Therefore, the substrate cannot bind effectively at the active site. So at low pH, the enzyme is protonated.
  • At point Y, which is the optimum pH. The substrate can bind effectively at the active site.
  • The enzyme is deprotonated at point Z, which is high in pH. It means it has a negative charge. Therefore, this substrate is enabled to bind effectively at the active site.

How pH affects the protons?

  • Increasing the ph level decreases the excess protons in the solution. It means deprotonating the solution.
  • When the ph level decrease, it adds excess protons to the solution. It means protonating the molecules that are there in the solution.

Experiments of the pH affect enzyme activity

Hydrogen peroxide is very toxic to both the body and the environment. It needs catalase to break it down into water and oxygen. Catalase is found in all organisms that breathe oxygen.

This enzyme facilitates the decomposition of hydrogen peroxide into water and oxygen, a toxic byproduct of the body.

The optimum pH for human catalase is approximately seven and has a fairly broad maximum. So the rate of reaction does not change too much at levels beyond like
6.8 and 7.5.

Sources of catalase include:

  • Sliced raw potato.
  • Ground meat.
  • Liver.

Hypothesis: When catalase, an enzyme, is added to a medium higher or lower pH outside its optimum pH range, the enzyme will denature. Therefore not perform its function of converting hydrogen peroxide into water and oxygen.


  • Hydrogen peroxide (3%).
  • Paper immersed with catalase.
  • pH 4, 7, and 10 solutions.
  • Beakers and a stopwatch.


  • Place the catalase paper at pH 4
  • Using a stopwatch to determine how long it takes for the reaction to take place.
  • Repeat steps 1-2 with catalase in solutions of pH 7 and pH 10.
  • Record results in a table and draw a graph of the results.


  • For pH 4, There was no reaction. The enzyme has denatured.
  • For pH 7, The Fastest reaction is at 90 seconds to complete.
  • For pH 10, the Slowest reaction is at 5 minutes to complete.

The pH 10 still achieved a reaction. The enzyme did perform at this pH level. The enzyme may be able to withstand varying pH levels. However, it performs best at pH 7 but not at pH 4.

Record all results in a table and draw a graph of the results.

Experiment of the pH affect enzyme activity
Experiments on the pH affect enzyme activity

The enzyme may be able to withstand varying pH levels. However, it performs best at pH 7 but not at pH 4. Each enzyme will work at a specific level of pH.

The Effect of pH on Enzymes


Stryer L, Berg JM, Tymoczko JL. Biochemistry (5th ed.). San Francisco: W.H. Freeman.
Murphy JM, Farhan H, Eyers PA. “Bio-Zombie: the rise of pseudoenzymes in biology.” Biochem Soc Trans.
Radzicka A, Wolfenden R. “A proficient enzyme”. Science.

Julia Rose

My name is Julia Rose. I'm a registered clinical therapist, researcher, and coach. I'm the author of this blog. There are also two authors: Dr. Monica Ciagne, a registered psychologist and motivational coach, and Douglas Jones, a university lecturer & science researcher.I would love to hear your opinion, question, suggestions, please let me know. We will try to help you.

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