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1.18: Enzymes and Allosteric Regulation

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    Catalysts and Enzymes

    Several criteria must be met for a chemical reaction to happen. Obviously, the reactants must first find one another in space. Chemicals in solutions don't "plan" these collisions, they happen at random. The rate (frequency of collisions per second) at which two reactants find one another will depend on their velocity (determined by temperature) and their concentration. In this (Biology) course, we'll assume temperature is a constant. Secondly, in addition to colliding, the molecules probably have to collide at the correct orientations, as not all collisions are potentially productive. Thirdly, the molecules have to have sufficient energy to form the transition state. If the transition state is significantly above the average energy of the molecules (which will be fairly uniform- a narrow distribution) very very few of the molecules will have sufficient energy to form the unstable, high tension, distorted "transition state". Thus, even if the reactants collide frequently, and the reaction is energetically favorable, a reaction with a activation energy significantly above the average energy of the reactants is not going to progress on a timescale suitable for the life of a cell. This is actually, and perhaps surprisingly, good news for the cell. It means that the cell can control metabolic flux by controlling the availability of catalysts.

    A catalyst has two qualities:

    It provides an alternate reaction path with low activation energy, and while it may participate in the reaction, it is not "used up" in the reaction (unlike the reactants and products).

    Although we and other biologists will often only consider one direction of a reaction, keep in mind that catalysts do not determine the direction of a reaction- they simply allow the reaction to occur in whichever direction is energetically favorable. Similarly, enzymes don’t change the ∆G of a reaction. In other words, they don’t change whether a reaction is exergonic (spontaneous) or endergonic. This is because they don’t change the free energy of the reactants or products. They only reduce the activation energy required to reach the transition state.


    Enzymes lower the activation energy of the reaction but do not change the free energy of the reaction. Here the solid line in the graph shows the energy required for reactants to turn into products without a catalyst. The dotted line shows the energy required using a catalyst.
    Attribution: Marc T. Facciotti (own work)

    Catalysts in biology are genetically encoded by the cell, and are called enzymes. Enzymes are made of protein(s), often with non-protein cofactors that are intimately involved in the actual reaction catalyzed (again, cofactors are part of the enzyme and are not "used up" in the reaction). There are some interesting exceptions in which the catalysis is actually performed by an RNA molecule, the structure of which may be stabilized by small proteins. These genetically-encoded catalysts are called "ribozymes" will be discussed in more detail later in the course.

    Section Overview

    Enzymes have an active site that provides a unique chemical environment, made up of certain amino acid R groups (residues) in a particular orientations and distance from one another. This unique environment is well-suited to convert particular chemical reactants for that enzyme, called substrates, into unstable intermediates (transition states). Enzymes and substrates are thought to bind with an "induced fit", which means that enzymes and substrates undergo slight conformational adjustments upon substrate contact, leading to binding. This subtle change in enzyme shape allows the enzyme to rapidly bind potential substrates in an "open" conformation" and then generate a tighter "closed" catalytically active alternative conformation only when the correct substrate is correctly aligned in the active site.

    Enzymes bind to substrates and can potentially catalyze reactions in four different ways (which might act together in a single enzyme): bringing substrates together in an optimal orientation, compromising the bond structures of substrates so that bonds can be more easily broken, providing optimal environmental conditions (often local pH) for a reaction to occur, and/or participating directly in their chemical reaction by forming transient covalent bonds with their substrates.

    Enzyme action must be regulated so that in a given cell at a given time, the desired reactions are being catalyzed and the undesired reactions are not. Inhibition and activation of enzymes via other molecules are important ways that enzymes are regulated. Inhibitors can act competitively or noncompetitively; noncompetitive inhibitors are usually allosteric (allo (other) steric (form). Activators can also enhance the function of enzymes allosterically. A common method by which cells regulate the enzymes in metabolic pathways is through feedback inhibition. During feedback inhibition, the products of a metabolic pathway serve as inhibitors (usually allosteric) of one or more of the enzymes (usually the first committed enzyme of the pathway) involved in the pathway that produces them.

    Enzyme Active Site and Substrate Specificity

    The chemical reactants to which an enzyme binds are the enzyme’s substrates. There may be one or more substrates, depending on the particular chemical reaction. In some reactions, a single-reactant substrate is broken down into multiple products. In others, two substrates may come together to create one larger molecule. Two reactants might also enter a reaction, both become modified, and leave the reaction as two products. The location within the enzyme where the substrate binds is called the enzyme’s active site. Since enzymes are proteins, there is a unique combination of amino acid R groups within the active site. Each amino acid side-chain is characterized by different properties. The unique combination of amino acids, their positions, sequences, structures, and properties, creates a very specific chemical environment within the active site. This specific environment is suited to bind, albeit briefly, to a specific chemical substrate (or substrates). Due to this jigsaw puzzle-like match between an enzyme and its substrates, enzymes can be extremely specific in their choice of substrates. The “best fit” between an enzyme and its substrates results from their respective shapes and the chemical complementarity of the functional groups on each binding partner.


    This is an enzyme with two different substrates bound in the active site (here conveniently squished down to 2 dimensions). The enzymes are represented as blobs, except for the active site which identifies three amino acids located in the active site (and shows the R group for one of them). The R group of R180 is interacting with the substrates through hydrogen bonding (represented as dashed lines), as are some groups in the peptide backbone. Amino acid positions are denoted a single letter code for the amino acid followed immediately by "position of the amino acid vs. the N terminal end". For example "R180" means an R (arginine) is the 180th amino acid from the N terminus. (Minor note- the R is this diagram is drawn incorrectly, though teh business end- where it connects to substrate- is OK.)

    At this point in the class you should be familiar with the chemical characteristics (charge, polarity, hydrophobicity) of the functional groups. For example, the R group of R180 in the enzyme depicted above is the amino acid Arginine (arginine's single letter code happens to be R, which is a little confusing in this context) and R180's R group consists of several "amino" functional groups. An amino functional group contains a nitrogen (N) and hydrogen (H) atoms. Nitrogen is more electronegative than hydrogen so the covalent bond between N-H is a polar covalent bond. The hydrogen atoms in this bond will have a partial positive charge, and the nitrogen atom will have a partial negative charge. This allows amino groups to form hydrogen bonds with other polar compounds. Likewise, the backbone carbonyl oxygens of Valine (V81) and Glycine (G121) the backbone amino hydrogen of V81 are depicted engaged in hydrogen bonds with the small molecule substrate.


    Look to see which atoms in the figure above are involved in the hydrogen bonds between the amino acid R groups and the substrate.
    Which substrate (the left or right one) do you think is more stable in the active site? Why? How?


    This is a depiction of an enzyme active site. Only the amino acids in the active site are drawn; the numbers refer to their positions in the primary sequence of the protein (and aren't really important here). The substrate is sitting directly in the center.
    Source: Created by Marc T. Facciotti (original work)


    First, identify the type of molecule in the center of the figure above. Second, draw in and label the appropriate interactions between the R groups and the substrate.

    Structural Instability of Enzymes

    The fact that active sites are so well-suited to provide specific environmental conditions also means that they are subject to influences by the local environment. It is true that increasing the environmental temperature generally increases reaction rates, enzyme-catalyzed or otherwise. However, increasing or decreasing the temperature outside of an optimal range can affect chemical bonds within the active site in such a way that they are less well suited to bind substrates. High temperatures will eventually cause enzymes, like some other biological molecules, to denature, a process that changes the natural properties of a substance. Likewise, the pH of the local environment can also affect enzyme function. Active site amino acid residues have their own acidic or basic properties that are optimal for catalysis. These residues are sensitive to changes in pH that can impair the way substrate molecules bind, because the charges on the R groups, and therefore both ionic and H-bonding interactions can change with pH. Enzymes are suited to function best within a certain pH range, and, as with temperature, extreme pH values (acidic or basic) of the environment can cause enzymes to denature.


    Enzymes have an optimal pH. The pH at which the enzyme is most active will be the pH where the active site R groups are protonated/deprotonated such that the substrate can enter the active site and the initial step in the reaction can begin. Some enzymes require a very low pH (acidic) to be completely active. In the human body, these enzymes are most likely located in the stomach, or located in lysosomes (a cellular organelle used to digest large compounds inside the cell).

    The process where enzymes denature usually starts with the unwinding of the tertiary structure through destabilization of the bonds holding the tertiary structure together. Hydrogen bonds, are ionic bonds easily disrupted by mild changes in temperate and pH, the disruption of covalent bonds would require more extreme conditions. Using the chart of enzyme activity and temperature below, make an energy story for the red enzyme. Explain what might be happening from temperature 37C to 95C.


    Enzymes have an optimal temperature. The temperature at which the enzyme is most active will usually be the temperature where the structure of the enzyme is stable or uncompromised. Some enzymes require a specific temperature to remain active and not denature.

    Induced Fit and Enzyme Function

    For many years, scientists thought that enzyme-substrate binding took place in a simple “lock-and-key” fashion. This model asserted that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view called induced fit. The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes a mild shift in the enzyme’s structure that confirms an more productive binding arrangement between the enzyme and the transition state of the substrate. This energetically favorable binding maximizes the enzyme’s ability to catalyze its reaction.

    When an enzyme binds its substrate, an enzyme-substrate complex is formed. This complex lowers the activation energy of the reaction and promotes its rapid progression in one of many ways. On a basic level, enzymes promote chemical reactions that involve more than one substrate by bringing the substrates together in an optimal orientation. The appropriate region (atoms and bonds) of one molecule is juxtaposed to the appropriate region of the other molecule with which it must react. Another way in which enzymes promote the reaction of their substrates is by creating an energetically favorable environment within the active site for the reaction to occur. Certain chemical reactions might proceed best in a slightly acidic or non-polar environment. The chemical properties that emerge from the particular arrangement of amino acid residues within an active site create the energetically favorable environment for an enzyme’s specific substrates to react.

    The activation energy required for many reactions includes the energy involved in slightly contorting chemical bonds so that they can more easily react. Enzymatic action can aid this process. The enzyme-substrate complex can lower the activation energy by contorting substrate molecules in such a way as to facilitate bond-breaking. Finally, enzymes can also lower activation energies by taking part in the chemical reaction itself. The amino acid residues can provide certain ions or chemical groups that actually form covalent bonds with substrate molecules as a necessary step of the reaction process. In this case, the enzyme is providing an alternate, lower-transition state energy path to the overall reaction. In all cases, it is important to remember that the enzyme will always return to its original state at the completion of the reaction. After an enzyme is done catalyzing a reaction, it releases its product(s). Always keep in mind that enzymes can also facilitate the reverse reaction. The net flux will depend on the reaction direction that provides a negative ∆G.


    According to the induced-fit model, both enzyme and substrate undergo dynamic conformational changes upon binding. The enzyme contorts the substrate into its transition state, thereby increasing the rate of the reaction.

    Creating an Energy story for the reaction above

    Using the figure above, answer the questions posed in the energy story.
    1. What are the reactants? What are the products?
    2. What work was accomplished by the enzyme?
    3. What state is the energy in initially? What state is the energy transformed into in the final state? This one might be tricky still, but try to identify where the energy is in the initial state and the final state.

    Speaking of energy: If a protein "bends" a substrate such that it approaches the transition state, where does the energy for that bending come from?

    Enzyme Regulation

    Why regulate enzymes?

    Cellular needs and conditions vary from cell to cell, and change within individual cells over time. The required enzymes and energetic demands of stomach cells are different from those of fat storage cells, skin cells, blood cells, and nerve cells. Furthermore, a digestive cell works much harder to process and break down nutrients during the time that closely follows a meal compared with many hours after a meal. As these cellular demands and conditions vary, so do the needed amounts and functionality of different enzymes.

    Regulation of Enzymes by Molecules

    Enzymes can be regulated in ways that either promote or reduce their activity. Although this inhibition might be the basis of the action of certain poisons, in many cases an enzyme may have evolved to respond to environmental influences (such as the concentration of relevant metabolites, which are not necessarily substrates or products) by regulating its own activity. There are many different kinds of molecules that inhibit or promote enzyme function, and various mechanisms exist for doing so. In some cases of enzyme inhibition, for example, an inhibitor molecule is similar enough to a substrate that it can bind to the active site and simply block the substrate from binding. When this happens, the enzyme is inhibited through competitive inhibition, because an inhibitor molecule competes with the substrate for active site binding. On the other hand, in noncompetitive inhibition, an inhibitor molecule binds to the enzyme in a location other than an active site. That binding alters the overall shape of the enzyme such that it no longer binds its substrate effectively. This type of inhibition is called allosteric inhibition.


    Competitive and noncompetitive inhibition affect the rate of reaction differently. Competitive inhibitors affect the initial rate but do not affect the maximal rate, whereas noncompetitive inhibitors affect the maximal rate.

    Discuss: Why are the effects of competitive inhibitors overcome by high concentrations of substrate?

    Most allosterically regulated enzymes are made up of more than one polypeptide, meaning that they have more than one protein subunit. When an allosteric inhibitor binds to an enzyme, all active sites on the protein subunits are changed slightly such that they bind their substrates with less efficiency. There are allosteric activators as well as inhibitors. Allosteric activators bind to locations on an enzyme away from the active site, inducing a conformational change that increases the affinity of the enzyme’s active site(s) for its substrate(s).


    Allosteric inhibitors modify the active site of the enzyme so that substrate binding is reduced or prevented. In contrast, allosteric activators modify the active site of the enzyme so that the affinity for the substrate increases.

    Video Link

    Check out this short (1 minute) video on competitive vs. noncompetitive enzymatic inhibition. Also, take a look at this video (1.2 minutes) on feedback inhibition.


    Many enzymes don’t work optimally, or even at all, unless bound to other specific non-protein helper molecules, either temporarily, through ionic or hydrogen bonds, or permanently through stronger covalent bonds. These helper molecules are termed cofactors. Binding to these molecules promotes optimal conformation and function for their respective enzymes. Cofactors may be inorganic ions such as iron (Fe2+) and magnesium (Mg2+), and these ions maybe be linked to larger nonprotein molecules. The term coenzyme is sometimes used to define a subclass of cofactors that are organic helper molecules, with a molecular structure made up of carbon, nitrogen and hydrogen, which are required for enzyme action (for example, a heme group, as a opposed to a metal ion or iron-sulfur cluster). There are also further specialized terms for subclasses of cofactors. These terms are employed loosely, variously, and irregularly by different scientists, and I suggest you stick with the safe, all-encompassing term "cofactor". The most common sources of organic cofactors are dietary vitamins. Vitamin C is a cofactor for multiple enzymes that take part in building the important connective tissue component, collagen. Hence a lack of vitamin C in our diet results in scurvy, a painful disease of connective tissue. An important step in the breakdown of glucose to yield energy is catalysis of pyruvate to acteyl coA by a multi-enzyme complex called pyruvate dehydrogenase. Pyruvate dehydrogenase is a complex of several enzymes that actually requires one inorganic cofactor (a magnesium ion) and five different organic coenzymes to catalyze its specific chemical reaction.

    Succinate dehydrogenase, an enzyme involved in both electron transport and the citric acid cycle, is an example of an enzyme that carries many cofactors, allowing it to transport electrons through the enzyme from the original donor molecule (succinate), through FAD/FADH (flavin), iron sulfur clusters, and heme, finally to Q, in the respiratory ETC. The function of this enzyme made possible only via the presence of various cofactors.


    Succinate dehydrogenase (SDH) oxidizes succinate to fumarate, using FAD as the immediate 2e- acceptor. The FAD of subunit SDHA is considered a cofactor as it does not leave the enzyme, but is directly oxidized by nearby iron sulfur clusters, within the B subunit of this enzyme. These are in turn oxidized by iron containing heme group within the SDHC and D subunits. Finally the electrons leave thsi complex via transfer to the membrane-diffusible molecule ubiquinone, also known as, coenzyme Q. The electrons will proceed further down respiratory ETC. All of these transfers, of course, can only occur if there is some electron acceptor at the end of the ETC.

    Enzyme Compartmentalization

    In eukaryotic cells, molecules such as enzymes are usually compartmentalized into different organelles. This allows for yet another level of regulation of enzyme activity. Enzymes required only for certain cellular processes can be housed separately along with their substrates, allowing for more efficient chemical reactions. Examples of this sort of enzyme regulation based on location and proximity include the enzymes involved in the latter stages of cellular respiration, which take place exclusively in the mitochondria, and the enzymes involved in the digestion of cellular debris and foreign materials, located within lysosomes.

    Additional Links

    Khan Academy

    The following links will take you to a series of videos on kinetics. The first link contains 4 videos on reaction rates and the second link contains 9 videos related to the relationship between reaction rates and concentration. These videos are supplemental and are provided to give you an outside resource to further explore enzyme kenetics.

    UCD Chemwiki

    Allosteric regulation

    1.18: Enzymes and Allosteric Regulation is shared under a not declared license and was authored, remixed, and/or curated by LibreTexts.

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