Background Informaion

Enzymes are a diverse and important class of proteins. Biologists refer to enzymes as biological catalysts because they increase the velocity or rate of chemical reactions in living cells. You may have already used MitochondriaLab to learn about basic principles of enzyme activity, metabolic pathways, and the role of enzymes in metabolism. Typically, each enzyme is capable of catalyzing a reaction between a very specific molecule or set of molecules. Molecules that an enzyme reacts with are called substrates.

Enzymes can catalyze very specific reactions for a given substrate with incredible precision and speed, in part because of the overall shape assumed by the amino acids that constitute the enzyme. Recall that the shape of any protein is typically influenced by the primary structure of the protein–the linear sequence of amino acids joined together by peptide bonds to form the protein. Enzymes also exhibit secondary and tertiary structure, and those with multiple subunits require quaternary structure for complete activity. Remember that these arrangements of protein structure determine the overall folding and shape or conformation of the enzyme, which in turn determines its function. There are few better examples of the important relationship between protein structure and function. All enzymes have a conformation that produces an active site–a pocket or groove in the enzyme where the substrate binds. The active site of an enzyme is typically specific for only one substrate, because the overall three-dimensional conformation of the active site is designed to fit the molecular shape of the substrate. For certain enzymes, the amino acids forming the active site and the way these amino acids interact with and bind to a substrate have been very well characterized. This is true for invertase, the enzyme you will study in this lab. Details about invertase will be discussed later in this background.

When enzymes were first studied, biochemists often compared the interaction between an enzyme and its substrate to the complementary interaction between a lock and key, where the enzyme and its active site represent the lock, while the substrate represents the key that fits the lock. Although this simple analogy may help to explain the specificity of an enzyme for a substrate, in reality this interaction is much more complex. Modern-day biochemists typically acknowledge that enzyme-substrate interactions follow an induced-fit hypothesis. This hypothesis states that the enzyme does not merely provide a static active site into which the substrate fits. Instead, as the substrate begins to enter the active site, the shape of the active site changes–induced by the substrate as it begins to enter the active site–thus enabling the enzyme to conform around the substrate and facilitate its binding to the active site.

Substrate binding to the active site of an enzyme is only the first step toward catalyzing a reaction. If enzymes function as biological catalysts, then how can we determine and measure the efficiency and rate of any reaction that a particular enzyme is carrying out on a given substrate that it binds? To begin, it is important to remember that one key aspect of enzyme activity is that while an enzyme speeds up the rate of a reaction, it is not altered by the reaction itself. An enzyme does not become part of the reactants or products. After an enzyme has catalyzed a reaction, it releases its substrate and then the active site of the enzyme is available to bind to another fresh substrate and repeat this process. For most enzymes this process, known as the catalytic cycle of enzyme activity, can be repeated very rapidly as long as there is enough substrate for the enzyme to react upon. Regardless of the type of reaction an enzyme is catalyzing–for example, a synthesis reaction or a degradation reaction–the catalytic cycle of an enzyme is often described by the following equation:

E + S --> ES --> E + P

This cycle begins when an enzyme (E) binds to a substrate (S) to form an enzyme-substrate complex (ES). Enzyme-substrate complexes typically form as a result of weak bonds between amino acids in the active site and atoms of the substrate. Depending on the type of reaction catalyzed by the enzyme, the enzyme then manipulates the substrate into the proper conformation to catalyze a reaction–for example, breaking bonds by hydrolysis, catalyzing the formation of new bonds, or rearranging atoms in the substrate to convert the substrate into a new molecule or molecules called products (P). Once the reaction has occurred, the enzyme releases the product(s); thus, the active site is free and available to bind to another substrate so the enzyme can repeat this cycle. Enzyme-catalyzed reactions can be reversible or irreversible.

Through this cycle, enzymes are said to lower the activation energy of a reaction–the energy required to make or break chemical bonds in a substrate to initiate a reaction. It is convenient to think about activation energy as a barrier that a cell must overcome to enable a reaction to take place. To understand why this is necessary for living cells, think about the factors that can regulate the rate of most reactions that you might carry out in a test tube in a chemistry lab. Increasing the temperature of the tube as well as increasing reactant concentration in the tube are two conditions that will increase the rate of most chemical reactions. Increasing temperature raises kinetic energy of reactants in the tube, thus increasing the likelihood of collisions between molecules. Raising reactant concentration results in more frequent collisions between molecules. Both factors typically increase the rate of a chemical reaction. Although heating or cooling a test tube is an effective way to regulate enzyme activity in a controlled laboratory environment, body temperature homeostasis prevents living organisms from raising and lowering body temperature to accommodate the large number of reactions that occur simultaneously in any given cell. Similarly, substrate concentrations for many biochemical reactions in a living cell are very low–in the nanomolar to picomolar range or lower–thus, it simply is not efficient for a cell to wait for kinetic energy to cause molecules to collide randomly and react with each other.

Think of enzymes as a set of "molecular hands" for a living cell. Consider the simple analogy of a bag of separate nuts and bolts. If screwing one nut onto one bolt is the reaction you want to carry out, you could expend a lot of energy shaking the bag until, over time, one nut randomly finds its way onto the end of a bolt! The energy you expended to begin to put a nut and bolt together is activation energy. Alternatively, you could speed up this reaction by reaching into the bag and using your hands to screw a nut onto a bolt, thus reducing the amount of energy required for this reaction to occur and greatly increasing the rate of this reaction.

Although enzymes are absolutely essential for accelerating biochemical reactions, a number of conditions influence enzyme activity. Enzymes don’t always operate at their maximal rate, however. Most enzymes demonstrate temperature and pH optimums–a temperature and pH at which enzyme activity is greatest. For example, as you might expect, blood enzymes perform optimally at a pH close to 7.4, the pH of normal human blood, whereas stomach enzymes have an optimal pH of around 2.0 to coincide with acidic conditions in the stomach. Varying these conditions typically affects the conformation of the enzyme, which in turn influences an enzyme’s ability to bind to its substrate and catalyze a reaction. Recall that when any protein unfolds, it becomes less active or inactive; this process is called protein denaturation. Enzymes can become denatured in response to an increase in temperature because raising temperature can break bonds–such as hydrogen bonds, Van der Waals attractions, and disulfide bridges–that are responsible for the secondary and tertiary structure of a protein. Changes in pH can also disrupt protein structure by changing hydrogen bond and ionic bond interactions, and by changing side group (R-group) interactions that contribute to the tertiary structure of an enzyme. In the most extreme circumstances, changing temperature or pH too dramatically–for example, by boiling–can completely denature an enzyme, causing it to lose all of its activity.

When biologists study enzyme-catalyzed reactions, we are typically interested in more detailed aspects of enzyme biochemistry than just substrates and temperature and pH optimums. When studying a particular enzyme, biologists often study the interactions between the enzyme and its substrate and the reaction rate of the enzyme in great detail. One of the most common approaches for measuring enzyme kinetics is the Michaelis—Menton equation, named after Leonor Michaelis and Maud Menten, two biochemists whose landmark discoveries in the early 1900s continue to serve as the basis by which important biochemical properties of enzyme kinetics are studied.

Once factor that strongly influences enzyme kinetics is the concentration of substrate [S] available for a particular enzyme. Consider the figure shown below. If we were to plot substrate concentration against initial reaction velocity (V or VO) as a measure of the rate of a reaction, many enzymes would show a pattern known as first-order kinetics. In first-order kinetics the rate of the reaction depends on the substrate concentration. Reaction rate increases as substrate concentration increases, because more substrate is available to be bound by the enzyme. If an enzyme were supplied with an infinite amount of substrate, then the reaction would reach a maximum velocity. This is because as the reaction proceeds, fresh substrate is rapidly binding to the active site of the enzyme, "saturating" all of the active sites for every enzyme molecule in the reaction. Under these conditions, adding additional substrate produces no effect on reaction velocity because the enzyme molecules are incapable of working any faster. This plateau of maximum velocity is abbreviated as Vmax.

Plotting [S] versus V for an enzyme-catalyzed reaction provides us with important information on the activity of the enzyme being studied because we can use Vmax to determine another important parameter of enzyme kinetics, the Michaelis constant (KM), which is a measure of the affinity of an enzyme for a substrate. The Michaelis constant is equal to [S] at -Vmax. To use EnzymeLab, you must be familiar with the basic factors involved in Michaelis—Menton kinetics as described in the previous two paragraphs. These factors are often expressed in the Michaelis—Menten rate equation as


VO =

KM + [S]

We can learn a great deal about enzyme activity from Michaelis—Menten measurements. In particular, KM is a measurement of the substrate concentration required for an efficient reaction to occur. The Michaelis—Menten equation can also be used to measure kcat, the turnover number, which tell us the catalytic ability of an enzyme to create product under saturated conditions. When biochemists are studying a reaction to determine if the enzyme involved follows Michaelis—Menten kinetics, data are typically plotted in one of two ways, as a Lineweaver–Burk plot or as an Eadie—Hofstee plot. These plots are created by rearranging the Michaelis—Menten equation. The Lineweaver—Burk equation is:







The Eadie—Hofstee equation is:

V = Vmax M



Examples of each type of plot are shown below.


In a Lineweaver—Burk plot, notice that the Michaelis equation is inverted to produce a plot with a straight line for V versus [S]. The slope of the line is KM/Vmax, the x-intercept tells us —1/KM, and the y-intercept is 1/Vmax. In an Eadie—Hofstee plot, V is plotted against V/[S]. The slope of the line tells us —KM, the y-intercept is Vmax, while the x-intercept is Vmax/KM.

Understanding the kinetics of individual enzymes is important for understanding the overall biochemistry of living cells. However, cell metabolism is dependent on the combined actions of many different enzymes that are essential for the anabolic and catabolic reactions that must occur to maintain the physiology of a cell. It is important to understand that many reactions that require enzymes rarely involve just a single enzyme that works by an all-or-none process. This would be like trying to manufacture a car from all of its components in one sweeping motion! Instead, many biochemical reactions involve cascades of enzymatic reactions, called metabolic pathways, that serve to catalyze a series of reactions. In a metabolic pathway, several enzymes work in a sequential fashion to convert reactants into a product or products. At each step in a metabolic pathway, intermediate molecules (metabolites) are produced that serve as the substrates for subsequent enzymes in the pathway. One benefit of a metabolic pathway compared to a single-enzyme reaction is that a cell can often precisely regulate the amount of product it can generate by independently controlling the catalytic activity of certain enzymes in the pathway.

Intermediate molecules are often an important part of the control of a metabolic pathway. One way in which metabolic pathways can be regulated by intermediates involves feedback inhibition. In this regulatory process, a final or end-product of a reaction can inhibit enzymes in the metabolic pathway. This process allows a cell to carefully control the amount of end-product that it produces as a way to prevent excess accumulation and waste of an end-product. Frequently, the end-product inhibits or blocks the activity of an enzyme at one of the initial, rate-limiting steps in the pathway to prevent the unnecessary production of intermediates. This would be similar to a car manufacturer, whose car supply has exceeded the public’s demand, stopping the auto assembly line at the first step rather than halfway through the assembly process to avoid producing half-completed cars.

For the purpose of studying cell metabolism, as well as for medical treatment and other applications, we can use molecules as enzyme inhibitors to artificially regulate enzyme activity. Some of these inhibitors can bind to enzymes in a reversible or an irreversible fashion. The two primary classes of enzyme inhibitors are called competitive and noncompetitive inhibitors. Competitive inhibitors are molecules that decrease an enzyme’s activity by binding to the active site of the enzyme and preventing binding of the enzyme’s normal substrate. Competitive inhibitors can work in this fashion because their molecular shape so closely resembles the natural substrate that the inhibitor is able to "compete" with the substrate for binding to the enzyme’s active site. Although competitive inhibitor molecules bind to the active site of an enzyme, catalysis does not occur.

Noncompetitive inhibitors also reduce or block an enzyme’s activity, but unlike competitive inhibitors these molecules do so by binding to a portion of the protein other than the active site. This binding results in a change in the overall three-dimensional conformation of the protein–altering the active site so that it will not bind to the substrate.

Because many enzymes are subject to inhibition by inhibitor molecules, biologists can take advantage of this property by designing compounds that can be used to modify enzyme activity for the purpose of treating human ailments and disease, as well as a number of other applications. For example, one of the more widely known competitive inhibitors is the antibiotic penicillin, which functions to block the active site of an enzyme required for cell wall synthesis by bacteria. Another example of a commonly used inhibitor is aspirin, which functions as a noncompetitive inhibitor of an enzyme involved in the production of prostaglandins, molecules that cause fever and inflammation. Last, two drugs employed in the treatment of acquired immunodeficiency syndrome (AIDS) function to inhibit enzymes in the human immunodeficiency virus (HIV). The first anti-HIV drug developed for patient use, AZT (3'-azido-2,'3'-dideoxythymidine), competitively inhibits an enzyme required for the synthesis of HIV DNA. Another class of enzymes, called protease inhibitors, function to reduce replication of HIV by competitive inhibition of an essential viral enzyme called HIV protease.

Now that you are familiar with some important biochemical properties of enzymes, it is time to put your knowledge to work. Countless numbers of different enzymes exhibit many of the properties discussed in this background. In EnzymeLab you will work with an enzyme that most likely played an important role in digesting some of the food molecules that you ate this morning for breakfast! The enzyme chosen for this lab is invertase (b -fructofuranosidase; E.C., also commonly called sucrase and saccharase. This enzyme uses water to catalyze the hydrolysis of the disaccharide sucrose. Sucrose, or "table sugar," is technically called a -D-glucopyranosyl (1 --> 2) b -D-fructofuranoside because it consists of the two simple sugars glucose (a -D-glucopyranosyl) and fructose (b -D-fructofuranoside) joined together by a glycosidic bond connecting carbon 1 of glucose to carbon 2 of the fructose molecule. Invertase cleaves the glycosidic bond between glucose and fructose. In animals, this reaction is required to digest fructose and release the monosaccharides glucose and fructose to make them readily available for absorption into the bloodstream. This reaction is important because most organisms cannot metabolize sucrose directly–it must first be converted to monosaccharides for cells to utilize sucrose as an energy source.

Invertase is present in a wide range of organisms including animals, plants, yeast, fungi, and algae. In humans, invertase is found on the surface of epithelial cells lining the inner walls of the small intestine. Depending on the source of invertase, the enzyme is active at a range of temperatures from 40° C to 70° C. Invertase can also be active in the pH range from 4.0 to 10.0. Two of your goals for this laboratory are to determine the optimal temperature and pH for the invertase you will study in EnzymeLab.

You will use EnzymeLab to study important biochemical parameters of invertase. You will set up an experiment by adding substrate to a test tube along with purified enzyme to determine temperature and pH optimums for invertase, to measure and calculate values of Michaelis—Menten kinetics, and to study the effect of inhibitors on invertase activity. Studying invertase will help you understand how biochemists determine the specific biochemical properties of an enzyme that follows Michaelis—Menten kinetics.


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  3. Schomburg, D., and Stephan, B., eds., Enzyme Handbook. Heidelberg, Germany: Springer-Verlag/Berlin, 1999.