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

Effect of pH on enzyme activity

In this lecture and experiment, we will be looking at the effects of pH on enzyme activity. In chemistry, pH calculates the relative proportions of hydrogen plus (H+) and hydroxide minus ions (OH−).

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

Enzymes speed up chemical reactions; to do this, they’ve got a groove on their surface called the active site. 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 rate of reaction 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, and as a result, increased changes from the optimum pH will result in the shape of the active site changes.

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?

The pH measures the acidity or basicity of a substance or a solution. Our stomach produces powerful acids that acid helps 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 foods we eat that are basic is baked beans, and we all know the results that 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 into more acidic conditions. Therefore the substrate can no longer bind, the amount of product form decreases, and the rate of reaction also decreases.

Results: The enzyme’s active site is natural if you increase or decrease the pH above or below the optimum pH. 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 very 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 about 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 its function is to best work 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 protein digestive 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, the liquid would pass through the body, and you wouldn’t be able to absorb the proteins and nutrients from the milk. A commercial form of rennin 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 affect 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.
  • At point Z, which is high pH, the enzyme is deprotonated. It means it has a negative charge. Therefore, this substrate is enabled to bind effectively at the active site.

How pH effects the protons?

  • When the ph level increases, it 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, a toxic byproduct of the body, into water and oxygen.

The optimum pH for human catalase is approximately 7 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 in 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 draws a graph of the results.


  • For pH 4, There was no reaction. The enzyme has denatured.
  • For pH 7, The Fastest reaction at 90 seconds to complete.
  • For pH 10, the Slowest reaction 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 the best at pH 7 and doesn’t perform 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 the best at pH 7 and doesn’t perform 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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