What are enzymes? Most enzymes are proteins. Proteins are polypeptides made up of amino acids. The polypeptides fold into secondary structures such as alpha helices and beta sheets. These secondary structures then fold into the protein’s tertiary structure. The tertiary structure of a protein is important for its function as an enzyme. When a protein unfolds, this structure is lost and the protein is no longer active.

Specific regions of the enzyme molecule bind other molecules called substrates. Substrates are the molecules that react. In this example, glucose and ATP are the enzyme’s substrates and the enzyme is hexokinase.

How do enzymes work? Enzymes catalyze reactions by lowering the activation energy. How do they lower the activation energy? Enzymes are relatively large molecules. They form many contact points with their substrates. The formation of the enzyme-substrate complex is the first step in an enzyme-catalyzed reaction.

The enzyme holds the substrate in the proper orientation for a reaction to occur. It places the substrate near specific amino acids and cofactors that help it react. The contact points or binding interactions between the enzyme and substrate result from hydrogen bonds, van der Waals interactions, and ionic interactions.

More enzyme-substrate binding interactions form once the substrate has started to react. This occurs at the top of the hill and is called an activated complex or transition state. When an enzyme binds a substrate, energy is released. The binding energy lowers the activation energy. Because the hill is lower, the reaction occurs more quickly.

Enzymes bind substrates and convert them to products. During the reaction, the enzymes are not changed. One enzyme can convert many substrate molecules into products. Now that we know what enzymes are and what they do, let’s learn more about how they do it.

SECTION_3: Enzyme Specificity

Enzymes are very selective in the types of reactions they catalyze and the types of substrates they act on. Let’s explore this concept of specificity by examining a biological process in which enzymes play a critical role: the pathway called glycolysis.

Glycolysis is a series of ten reactions that convert glucose to pyruvate. Pyruvate is an intermediate formed when glucose is metabolized to carbon dioxide and water.

How do enzymes in this metabolic pathway know which reactions to catalyze? Let’s focus on the first reaction in the glycolysis pathway. The enzyme hexokinase adds a phosphate to glucose. It doesn’t add the phosphate just anywhere. It specifically adds it to the C-6 hydroxyl. Other enzymes exist that phosphorylate the other hydroxyl groups.

Hexokinase adds the phosphate to the C-6 hydroxyl because of the way the enzyme specifically binds the glucose molecule. Hexokinase can’t phosphorylate glucose directly because the free energy change is positive, so it couples glucose phosphorylation to ATP hydrolysis. To couple these reactions, hexokinase has a binding site for ATP and another for glucose. This is the enzyme’s active site, where catalysis occurs. Catalysis is the change in the reaction rate by a catalyst.

Because ATP hydrolysis is spontaneous, it doesn’t need the glucose in order to react. It can react with water to form ADP. Water is smaller than glucose and easily enters the active site. Reacting ATP with water is like setting dollar bills on fire. You get a little bit of heat but nothing else. Hexokinase must specifically catalyze the reaction of ATP with glucose and no other molecule.

How does this occur? Hexokinase binds ATP only after glucose binds. This prevents the enzyme from catalyzing the reaction of ATP with water. The enzyme changes its shape after glucose binds. This is called induced fit. The shape change promotes ATP binding, thereby improving the enzymatic activity.

The many points of contact between the enzyme and its substrate also ensure specificity. Molecules with slightly different shapes won’t fit into the active site as well as the substrate. This reduces the ability of the substrate to interact with the enzyme and the activation energy remains high.

Often an enzyme can’t do its job alone. Molecules called cofactors are needed. A cofactor is a molecule required for the action of certain enzymes. Cofactors may be inorganic ions, such as iron or magnesium. The enzyme hexokinase requires a magnesium ion as a cofactor. Magnesium ions function to shield the negatively charged groups of ATP and facilitate the reaction with glucose.

Coenzymes are also cofactors. A coenzyme is an organic molecule often derived from vitamins. Unlike an enzyme, a coenzyme may be modified in a reaction. The coenzymes NAD and NADP carry hydride ions in oxidation-reduction reactions. A hydride ion is equivalent to a proton and two electrons.

Enzymes are the world’s best catalysts. Industrial catalysts speed up reactions by 3 to 5 orders of magnitude. They often require high temperatures, and they aren’t very specific. Biological catalysts increase reaction rates by 9 to 15 orders of magnitude. They work at room temperature and are extremely specific.

In the next section, we’ll learn more about reaction rates when we discuss enzyme kinetics.

SECTION_4: Enzyme Kinetics

Enzyme kinetics describe how reaction rates are measured. We can use enzyme kinetics to determine how long it takes an enzyme to convert a substrate into a product.

Let’s look at the conversion of hydrogen peroxide to water and molecular oxygen. Hydrogen peroxide is a strong oxidizer. It’s used to clean scrapes and cuts. A bottle stored in a medicine cabinet lasts for a year or longer.

Inorganic catalysts, such as iodide ions speed up the reaction rate by about 5 orders of magnitude. Let’s do an experiment to see how the catalyst works. Add some hydrogen peroxide and a little soap to a graduated cylinder by dragging them to the cylinder. First, drag the soap to the cylinder and then click the play button. Note that there is some liquid in the cylinder. Now drag the hydrogen peroxide to the cylinder and click the play button again. Note that there is more liquid in the cylinder. Now add the catalyst potassium iodide by dragging it to the graduated cylinder. Click the play button: See all the bubbles? They result from the oxygen produced in the reaction.From SME: Instructions are unclear: Instructions state to drag hydrogen peroxide and soap to cylinder but are not clear about clicking the play button after dragging each bottle to the cylinder...the liquids do "appear" in the cylinder if the play button is clicked after the liquids are dragged. It does bubble when all 3 of the substances are in the cylinder and the play button is clicked. I found this frustrating/annoying to use because of lack of instructions and I suspect that some students will also. Perhaps the instructions can be clarified! I placed some suggestions in the script.JAE: Yes, they do need to be clarified...they make no sense as they are now. Maybe we can add some text on screen (rather than any VO) to help clarify? See me about this one

From SME:

Instructions are unclear: Instructions state to drag hydrogen peroxide and soap to cylinder but are not clear about clicking the play button after dragging each bottle to the cylinder...the liquids do "appear" in the cylinder if the play button is clicked after the liquids are dragged. It does bubble when all 3 of the substances are in the cylinder and the play button is clicked. I found this frustrating/annoying to use because of lack of instructions and I suspect that some students will also. Perhaps the instructions can be clarified! I placed some suggestions in the script.

JAE: Yes, they do need to be clarified...they make no sense as they are now. Maybe we can add some text on screen (rather than any VO) to help clarify? See me about this one

Ever notice how hydrogen peroxide also bubbles when you pour it on a cut? A catalyst must be present. The enzyme catalase is released when cells are damaged. Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen. Catalase has four subunits, just like hemoglobin, and an iron- containing heme group. It also requires the coenzyme NADP. Catalase speeds up the decomposition of hydrogen peroxide by 15 orders of magnitude! Catalase can degrade 10 million molecules of hydrogen peroxide in 1 second.

The reaction rate depends on how often an enzyme and substrate collide. The catalase reaction is limited by how fast the reacting molecules can diffuse and collide with the enzyme. Reaction rates depend on the reaction conditions. Since a reaction can’t occur any faster than the time it takes for two molecules to collide, reaction rates are sensitive to concentration. The rate of an enzyme-catalyzed reaction will increase with concentration until the enzyme is saturated with substrate. When the enzyme is saturated, all active sites are filled with substrate.

In general, only initial rates are used in kinetic calculations. The initial rate can be measured in this region of the left graph, which shows change in substrate concentration as a function of time. This is important because the substrate concentration decreases as the reaction proceeds, thus affecting the reaction rates whenever the enzyme is not saturated.

Temperature also affects reaction rates. In general, the rate of a reaction increases as temperature increases. Recall the combustion of sugar. However, the reaction rates of enzyme-catalyzed reactions won’t keep increasing forever. At high temperatures, proteins unfold and denature. Because an enzyme’s catalytic ability depends on its structure and being able to form lots of substrate binding interactions, the unfolded enzyme doesn’t work very well.

Many of the reactions that enzymes catalyze involve the transfer of protons. Proton transfer reactions are very sensitive to pH. Recall that pH is a function of proton concentration in the solution. A protein’s structure is sensitive to pH. Extremes in pH cause proteins to unfold. Each enzyme has its own unique temperature and pH profile.

Because of the charges on substrates and enzymes, ion concentrations influence reaction rates. The ions shield the charges to prevent proteins from aggregating and precipitating out of solution.

In addition to environmental conditions, molecules called activators and inhibitors affect enzyme activity. Activators and inhibitors are molecules that bind to enzymes. Activators and inhibitors are like the volume controls on a stereo. Activators turn up the volume and improve the catalytic ability of enzymes; inhibitors turn the volume and the enzyme’s activity down or off. Some activators and inhibitors change an enzyme’s shape. An enzyme can have conformations with different activities. Recall how hexokinase’s shape changed upon glucose binding.

Let’s look at a dimeric enzyme that has two forms, an active form and an inactive form. When an inhibitor binds, the inactive form is favored. When an activator binds, the active form is more likely. Many metabolic pathways are regulated in this way. This is called allosteric regulation. These molecules, called effectors, bind at a site other than the active site. Another type of an inhibitor is a competitive inhibitor. Competitive inhibitors compete with the enzyme’s substrate for the active site.

An enzyme’s activity depends on its structure. Its structure depends on temperature, pH, ion concentration, and effector concentration. Enzyme activity also depends on substrate concentration and the concentration of competitive inhibitors. Many drugs exert their therapeutic effects by acting as competitive inhibitors of target enzymes.

SECTION_5: Summary

Enzymes are the world’s best catalysts. They work in water at relatively low temperatures and with extreme specificity. They cannot make an unfavorable reaction spontaneous, nor can they change how far a reaction proceeds. They can lower the energy barrier, which speeds up the reaction rate.

Enzyme kinetics describe enzyme-catalyzed reaction rates. Energy is released when an enzyme binds its substrate. This energy is used to lower the activation energy. The lowering of the activation energy enables the reaction to proceed faster. This is the basis of enzyme catalysis.

An enzyme maximizes binding interactions and binding energy through induced fit. Induced fit also promotes enzyme specificity.

Enzymes speed up reactions. They increase the reaction rate. A reaction rate is a change in concentration as a function of time. Reaction rates are influenced by substrate concentration, temperature, pH and ion concentration. Rates are also influenced by the presence of activators and inhibitors. Many drugs act as competitive inhibitors. Some HIV protease inhibitors act in this way.

It’s important to understand biological catalysts, because they're central to all metabolic reactions. We would not be alive without them!

Copyright 2006 The Regents of the University of California and Monterey Institute for Technology and Education