Peptides, Proteins, and Enzymes, Exams of Biochemistry

Download Peptides, Proteins, and Enzymes and more Exams Biochemistry in PDF only on Docsity! 1 Chapter 13 Lecture Notes: Peptides, Proteins, and Enzymes Educational Goals 1. Describe the general bonding pattern of α-amino acids and understand how amino acids are classified by the polarity and charge of their side-chains. 2. Given the table of twenty common amino acids, determine the total charge of the dominant form of an amino acid (at physiological pH, at pH < 2, and at pH > 11). 3. Understand the three-dimensional information contained in the Fischer projection of an amino acid. 4. Given a Fischer projection of an amino acid, determine if it is an L-amino acid or D-amino acid. 5. Understand and define the term peptide. 6. Given the table of twenty common amino acids, be able to draw the structural formula of the peptide that is obtained when two or more particular amino acids are connected by peptide bonds. 7. Given the structural formula of a peptide be able to identify the peptide bonds, the C-terminus, the N-terminus, and the peptide groups. 8. Given the structural formula of a peptide and the table of twenty common amino acids, determine the total charge of the peptide’s dominant form (at physiological pH, at pH < 2, and at pH > 11). 9. Given the structure of a peptide, and the table of twenty common amino acids name a particular peptide using amino acid residue abbreviations. 10. Compare and contrast peptides and proteins. 11. Understand and define primary, secondary, tertiary, and quaternary protein structure. Name the noncovalent interactions that are responsible for each level of structure. 12. Explain what is meant by the term denaturatio and list the ways to denature a protein. 13. Understand the difference between globular, fibrous, and membrane proteins. 14. Compare and contrast simple proteins and conjugated proteins. 15. Understand the terms cofactor and coenzyme. 16. Understand how enzymes work and distinguish between absolute specificity, relative specificity, and stereospecificity. 17. Understand and define the terms: essential amino acid, complete protein, incomplete protein, and complimentary protein. 18. Understand how changes in pH and temperature can affect the reaction rate of an enzymatically- catalyzed reaction. 19. Understand how enzyme inhibitors and activators control enzymatic reactions, and compare and contrast reversible and irreversible inhibitors. 20. Understand how organisms regulate metabolic pathways using feedback inhibition and positive feedback. 2 Introduction The ___________ ___________________of amino acids in a protein and the chemical nature of the amino acid __________ _________________ enable proteins to perform their functions. • Typical protein functions: § Catalyze Reactions (enzymes) § Chemical Signaling (hormones) § Storage (e.g. myoglobin stores oxygen) § Structural (e.g. collagen in skin and tendons) § Protective (e.g. antibodies) § Contractile (e.g. myosin in muscle) § Transport (e.g. hemoglobin) Amino Acids Structure of Amino Acids Amino acids are organic compounds that contain a and a . For amino acids, the R-group is often called the “side-chain” or “variant group.” The side-chain can be a hydrogen atom, hydrocarbon, or various other groups of bonded atoms. Amino acids are named based on the identity of their . • For example, if the side-chain is a hydrogen atom (H), then the amino acid is called glycine; if the side-chain is a methyl group (CH3), then the amino acid is called alanine. There are 23 amino acids that make up the proteins in plants and animals, 20 of them are directly specified by the genetic code in DNA. These twenty amino acids are called the amino acids. • All twenty common amino acids are amino acids. • They are called α-amino acids because their side-chains are attached to α-carbons. REMINDER: The α-carbon is the carbon that is bonded to the carboxyl group’s carbonyl carbon. 5 The predominant form of alanine has a negative (1-) formal charge on the carboxylate group and a positive (1+) formal charge on the quaternary ammonium group, which gives it a total charge of . predominant form of alanine at pH = 7.4 When an amino acid has a total charge equal to zero, it is called a . • (zwitter is German for hermaphrodite or hybrid). The amino acid structures in the table (provided earlier) are the predominant forms at physiological pH. In sufficiently acidic or basic solutions, the of the predominant form of an amino acid will change from its physiological value. EXAMPLE: Consider the total charge of the predominant form of alanine in an extremely acidic solution. At pH = 1.0 (an extremely acidic solution) the pH is than the pKa of both the carboxyl group and the quaternary ammonium group, therefore both groups exist in their acid form, as shown below. predominant form of alanine at pH = 1.0 The predominant form of alanine at pH = 1.0 has an uncharged carboxyl group (COOH) and has a positive (1+) formal charge on the nitrogen of the quaternary ammonium group, which results in a _________________ (1+) total charge. EXAMPLE: Consider the total charge of the predominant form of alanine in an extremely basic solution. At pH = 12.0 (an extremely basic solution) the pH is than the pKa of both the carboxyl group and the quaternary ammonium group, therefore both groups exist in their base form, as shown below. predominant form of alanine at pH = 12.0 The predominant form of alanine at pH = 12.0 has a negative (1-) formal charge on the single-bonded oxygen of the carboxylate group and an uncharged nitrogen in the amine group, which results in a _________________ (1-) total charge. 6 Practice Problems: The amino acid structures in the table provided earlier are the predominant forms at physiological pH. a. Draw the predominant form of valine when the pH = 7.4 b. Draw the predominant form of valine when the pH = 1.0 c. Draw the predominant form of valine when the pH = 12.0 d. What is the total charge of the predominant form of valine when the pH = 7.4? e. What is the total charge of the predominant form of valine when the pH = 1.0? f. What is the total charge of the predominant form of valine when the pH = 12.0? Classification of Amino Acids Amino acids are classified by the of their side-chain and the ability of their side-chain to acquire (at physiological pH). Amino Acid Class Side Chain Polarity Side-Chain Charge at Physiological pH Nonpolar nonpolar (hydrophobic side-chain) zero Polar neutral polar (hydrophilic side-chain) zero Polar acidic polar (hydrophilic side-chain) negative Polar basic polar (hydrophilic side-chain) positive 7 1) Nonpolar Amino Acids Nonpolar amino acids have nonpolar (hydrophobic) side-chains and their predominant forms have uncharged side-chains at physiological pH. • The nonpolar amino acids (their predominant forms at physiological pH) are: Note that although the side-chain of tryptophan contains a few highly-polar bonds, the hydrocarbon part is so large that it dominates the interactions, making the side-chain hydrophobic. For this reason, tryptophan is put into the nonpolar class. 2) Polar Neutral Amino Acids Polar neutral amino acids have polar (hydrophilic) side-chains and their predominant forms have uncharged side-chains at physiological pH. • The polar neutral amino acids (their predominant forms at physiological pH) are: 10 Peptides and Proteins The Peptide Bond Peptides and proteins consist of amino acid residues joined by ____________________ (amide) bonds. Formation of a Peptide Bond Step 1: The two amino acids are drawn side-by-side. The single-bonded oxygen atom is removed from the carboxylate group on the left-most amino acid. Two hydrogen atoms are removed from the quaternary ammonium group on the right- most amino acid. The oxygen atom and the two hydrogen atoms combine to form a water molecule. Step 2: A new bond is made between the carbonyl carbon and the nitrogen. The peptide formed in this example is called a because it contains two amino acid residues. The new bond between the two amino acid residues is called a peptide bond. . You try one: Draw the structural formula of the dipeptide that contains two valine amino acid residues. Label the peptide bond 11 Formation of Larger Peptides Larger peptides are formed by adding more amino acids, one by one, to a growing peptide. Example: Formation of a Tripeptide • Begin with the general form of a dipeptide and then add a new amino acid residue. • The new peptide bond can be made using the same two steps as we used when we made a dipeptide. This process can continue and larger peptides can be formed by adding more amino acids, one by one, to a growing peptide. Peptide Terminology The end of the peptide structural formula that has a quaternary ammonium group is called the ____-terminus, and the end that has a carboxylate group is called the __________-terminus. The bonding pattern around a peptide bond is called the peptide _____________. Note that nitrogen in a peptide group does not have a (1+) formal charge, as does the nitrogen in the quaternary ammonium group at the N-terminus. 12 Peptides are identified by the use of a common name or, by listing its amino acid residues’ three-letter abbreviations in order from N-terminus to C-terminus. Example of identifying a peptide from its amino acid residue’s abbreviations: Val-Asp-Ala-Arg-Gly. I drew this pentapeptide by forming peptide bonds between the predominant forms of the amino acids at physiological pH, therefore the resulting pentapeptide is also in the form that is predominant at physiological pH. Note that two of the side-chains in this peptide carry a formal charge. This peptide has a total charge equal to zero because the two negative charges and two positive charges add up to zero. You try one: a. Draw the structural formula for the predominant form of Gly-Lys-Tyr-Ala at physiological pH. b. Label the peptide bonds and circle the peptide groups. NOTE: If you correctly connect the amino acid structural formulas from the amino acid table, then the peptide that you draw will be the predominant form at physiological pH. Also: What is the total charge of the peptide that you drew for in the previous problem? 15 The Beta Sheet The beta sheet geometry occurs when a peptide folds back on itself in a ________________________ arrangement. Illustrative Model of a Beta Sheet In addition to alpha helices and beta sheets, there are a few other, much less frequently seen geometries that are also categorized as secondary structures. Since these other secondary structures are relatively rare, I will not discuss their particularities. A key feature of secondary protein structure is that it only involves hydrogen bonding between peptide groups within an individual peptide chain. Tertiary Protein Structure Alpha helices and/or beta sheets, along with the unorganized sections of a peptide chain, “fold” into a more compact shape. • The shape of a peptide is called the tertiary structure. “Ribbon models” are often used in order to visualize tertiary protein structure. These illustrative models use ribbon-like shapes to represent the geometry of secondary structures. The spring-like ribbons represent alpha helices and the flat side-by side ribbons represent beta sheets. Sometimes arrows are used at the ends of ribbons to indicate the direction (from N-terminus to C-terminus). Lines or thin tubes are used for unorganized sections of a peptide chain. The ribbon model for ribonuclease A protein (RNase A), an enzyme used to break down RNA, is shown on the right. Ribbon Model of an RNase A Protein Source: Wikimedia Commons, Author: Vossman, CC-BY-SA, http://creativecommons.org/ licenses/by-sa/2.5/deed.en 16 Of the many folding patterns (conformations) possible for a protein, there is usually only one that leads to a _______________________(biologically active) molecule. The sequence of amino acids (primary structure) ultimately determines which folding pattern is selected, so both secondary and tertiary structure ______________________on primary structure. Some of the interactions that are involved in tertiary structure are illustrated below. Description of Tertiary Structure Interactions: 1) Hydrophobic Interactions Nonpolar side-chains are attracted to other nonpolar side-chains through London forces, and form “water-free pockets” in the interior region of the folded and compacted peptide (see the illustration above). 2) Hydrogen Bonding Hydrogen bonding in tertiary structures can occur between polar side chains (that contain the features necessary for hydrogen bonding) and/or peptide groups. See the illustration above. 3) Salt Bridges I introduced salt bridges to you, in chapter 4, as one of the five noncovalent interactions. A salt bridge is an attractive force between the positive formal charge on polar basic amino acid residue and a negative formal charge on a polar acidic residue (see the example in the illustration above). 17 4) Disulfide Bridges In a previous chapter, you learned that disulfide (covalent) bonds can be formed by the oxidation of two thiol (SH) groups. Disulfide bonds in proteins are called disulfide bridges. Each cysteine residue contains a thiol group in its side-chain that is capable of forming a disulfide bridge with another cysteine residue, as shown above. 5) Dipole-Dipole and Ion-Dipole Forces Dipole-dipole attractive forces can occur between polar side-chains and/or peptide groups. These interactions are not included in the illustration on the previous page. If needed, you can review dipole- dipole and ion-dipole interactions in section 6 of chapter 4. Quaternary Structure A large number of native proteins are a combination of ______ _________ ____________ polypeptide chain. • Example: Hemoglobin Quaternary protein structure is the overall shape that occurs when two or more ____________ peptide chains assemble to make a protein. In proteins composed of two or more peptide chains, the individual peptide chains are referred to as “subunits.” The quaternary structures of large proteins are sometimes depicted using space-filling models. In these models, the various subunits are often shaded with different colors or grey-scale tones. • Example: ATP synthase The forces that hold the subunits together in quaternary structures are the same as those involved in tertiary structures. Image source: Wikimedia Commons, Author: Richard Wheeler, CC-BY-SA, http://creativecommons.org/licenses/by- sa/3.0/legalcode Image source: Wikimedia Commons, Author: Alex.X CC-BY-SA, http://creativecommons.org/licenses/by- sa/3.0/legalcod 20 The immune system can produce an almost infinite variety of paratope shapes by varying the paratope region’s amino acid sequence (and therefore its shape). By doing so, antibodies are produced to be specific for one particular antigen, much like a lock is specific for one key. Some antibodies contain more than one immunoglobulin unit. Placental mammals, which includes humans, have immunoglobulin monomer, immunoglobulin dimer, and immunoglobulin pentamer antibodies. Immunoglobulin dimers are made from two immunoglobulin monomers, and immunoglobulin pentamers are made from five immunoglobulin monomers. These three types of antibody structures are illustrated below. One last note on antibodies: Antibodies have (oligosaccharides) that are covalently bound to some of their amino acid residue side-chains. • Proteins, such as antibodies, that contain carbohydrates are called __________________. Fibrous Proteins Fibrous proteins have long and narrow “_______________ -like” shapes. • They are much less compact than globular proteins. The narrower shape makes it difficult for hydrophobic side-chains to be oriented toward the interior region of a fibrous protein, and results in a hydrophobic exterior. For this reason, fibrous proteins tend to be water-insoluble. Fibrous proteins play important roles in providing structural rigidity and in contractile movement (muscles). 21 An example of a fibrous protein is collagen. Collagen is the most abundant protein in the body. Its function is to provide structural rigidity and stiffness. It is found in skin, ligaments, tendons, and other parts of the body. An illustration of the components of collagen are shown below. Other examples of fibrous proteins are keratins. Their primary role is to provide structural rigidity and stiffness. Keratins are some of the strongest natural materials. • Keratins can be classified as alpha-keratins or beta-keratins. Alpha-keratins are found in places such as hair, wool, horns, hooves, claws, and nails. In hair, two peptide double helices are twisted around each other to form a protofibril, as shown below. Protofibrils bundle together to form microfibrils. Microfibrils bundle together to form macrofibrils. Each hair cell is primarily composed of bundled macrofibrils. A single hair consists of bundled hair _______________. 22 Beta-keratins Beta-keratins, which are also fibrous proteins, are found in places such as reptilian skin, the outer layer of human skin, bird feathers and beaks, turtle shells, silk, and the tongue. • Beta-keratins are composed of fibers that primarily contain beta sheet secondary structures. • The beta sheets are stacked in ____________________ tertiary structures. An example of a beta-keratin structure can be seen in silk. The stacked beta sheets, which are held together by disulfide bridges and noncovalent interactions, entwine to form a fibroin microfibril. Fibroin microfibrils assemble to form fibrils. Fibrils assemble to form fibroin filaments. Two fibroin filaments are held together by sericin protein, which acts like a glue to hold the two fibroin filaments together in a single silk fiber, as shown below. Fibroin microfibril keratin fibers are also found in spider webs. The structure of spider’s silk is illustrated below. 25 1) Actin contains sites to which myosin heads can bind. In our initial state, tropomyosin fibers block actin’s myosin binding sites so that the myosin heads are unable to attach to the thin filament, as shown in the illustration on the right. 2) Muscle contraction begins in response to an action potential (nerve impulse) that originates in the central nervous system. • The electrical signal is transferred to a particular muscle and causes an organelle called the sarcoplasmic reticulum to release calcium ions. • When calcium ions are released, they bind to troponin, which causes the tropomyosin fibers to move and thereby exposes the myosin binding sites. 3) ATP is hydrolyzed to ADP and Pi. Energy released from the hydrolysis of ATP reaction is used to change the conformation (shape) of myosin. This results in a “cocked” myosin head. • This is analogous to “cocking the hammer” of a pistol, or pulling back on the string of a bow-and-arrow. In this step, the ADP and Pi that are produced remain attached to the “cocked” myosin head, as shown on the right. 4) The “cocked” myosin head attaches to a myosin binding site on the thin filament. This attachment is a noncovalent interaction. 26 5) ADP and Pi are released from the myosin head. This allows the myosin to bend back to its original “un-cocked” position. • In our “cocked” pistol analogy, this step represents what happens when the trigger of a pistol is pulled: the pistol’s “hammer” springs forward (to strike the bullet’s cartridge). • In our bow-and-arrow analogy, this step represents what happens when the string is released: it moves forward and accelerates the arrow. Because the myosin head is attached to the thin filament, as the myosin bends, the thin filament “slides” past the thick filament. 6) ATP binds to the myosin head, which causes the head to detach from the thin filament. This completes the cycle; the system is now back to its original configuration and the cycle can repeat so long as calcium and ATP are present. As this cycle repeats, the muscle can continue to shorten. Since calcium ions are constantly being transported back into the sarcoplasmic reticulum, their release must be continuously induced by central nervous system impulses in order for muscle contraction to continue. If ATP is not present, the myosin remains bound to the thin filament. This state is observed after death, since ATP is no longer produced, and is called rigor mortis. 27 Membrane Proteins Membrane proteins are proteins that are ________________ to biological membranes. Membrane proteins function as enzymes, cell recognition markers, receptors (allowing chemical signals to be relayed between the interior and exterior of cells), and transporters of compounds in and out of cells. Some membrane proteins extend through the __________________ membrane and are called transmembrane proteins. Examples of transmembrane proteins include the aquaporins. • Aquaporins function as transporter proteins; they facilitate the transport of water molecules (only) in and out of cells. There are several types of aquaporins, one of them, aquaporin-1, is illustrated on the right. Some membrane proteins do not completely extend through the membrane; these are called __________________ proteins. An example of a monotopic protein is cyclooxygenase-2. • Cyclooxygenase-2 is responsible for converting eicosanoic acid into prostoglandins, prostoscyclin, and thromboxane (you learned about this enzyme and these reactions in a previous chapter). An illustration of cyclooxygenase-2 attached to a membrane is shown on the right. Source: The protein structure is from Wikimedia Commons, Author: Vossman CC-BY-SA, http://creativecommons.org/licenses/by-sa/3.0/legalcode Understanding Check: Globular vs. Fibrous vs. Membrane Proteins Do a bit of online research to determine if succinate dehydrogenase is a globular, fibrous, or membrane protein. 30 Foods that contain proteins but do not contain all of the essential amino acids are called ______________ proteins. • These include most plant proteins. o Examples of incomplete proteins and their missing essential amino acids are listed in on the right: Combining of two or more incomplete proteins that are deficient in different amino acids is a dietary strategy used to ensure the intake of all nine essential amino acids. • For example, if you eat beans and rice, you obtain all of the essential amino acids since rice contains the amino acids that beans lack, and vice versa. • When proteins are combined in this way, they are called _______________________ proteins. Enzymes Catalysts are substances that increase the rates of chemical reactions. Life requires that many chemical reactions occur within organisms. The human body employs over a thousand chemical reactions. Many of these reactions would occur too slowly to be useful in the absence of a catalyst. Nature provides humans and other biological organisms with proteins that are capable of catalyzing reactions. Protein catalysts are called ___________________. • Among all plants and animal species, over 5,000 chemical reactions are catalyzed by enzymes. • Enzymes are capable of increasing the rate of a chemical reaction by up to a factor of one thousand. Scientists who specialize in studying enzymes are called enzymologists. Enzymologists refer to the reactants of catalyzed reactions as ___________________. • Most enzymes are composed of hundreds or thousands of amino acid residues, however only a small region of the enzyme makes contact with the substrates. Let’s take a look at a model that describes enzymatic catalysis. The part of the enzyme that makes contact with substrates is called the “____________ ____________.” In this model, we will represent an enzyme and its active site as illustrated on the right. Food Amino Acid Deficiency rice, wheat, oats lysine beans methionine, tryptophan peas methionine soy low in methionine corn lysine, tryptophan almonds, walnuts lysine, tryptophan Understanding Check Which two foods (from the table above) could each be eaten with corn as a complementary protein? 31 We will consider a reaction where two substrates (reactants) are converted to one product, as illustrated below. In this example, two substrates react to form one product, however this model will also apply to other cases such as one substrate compound forming two products, or two substrate compounds forming two products. The enzymatic catalysis model is illustrated below. In Step 1, the substrates bind to the active site of an enzyme. • The substrates are held tightly in the active site by noncovalent attractive forces, which are maximized due to the complementary shapes of the substrates and active site. • The particle that is formed when the substrates are bound to the enzyme is called the enzyme- substrate complex. In Step 2, the chemical reaction occurs. • Substrates are converted to products when covalent bonds within the substrates are broken and/or new bonds are made. • The particle that is formed when the product is bound to the enzyme is called the enzyme-product complex. In Step 3, the newly formed product is released. • Note that after products are released from enzymes, the enzymes are free to accept new substrates and the cycle can repeat. Enzymes ____ _______ affect the equilibrium concentrations of products and reactants (substrates), they only increase the reaction rates, and therefore equilibrium is reached more quickly. 32 All catalysts, including enzymes, ________________ the activation energy of a reaction and thereby increase the reaction rate. Compare the reaction energy diagram of an enzymatically catalyzed reaction with that of an un-catalyzed reaction: The un-catalyzed reaction is represented by the solid curve and the enzymatically catalyzed reaction is represented by the dashed curve. In catalyzed and un-catalyzed reactions, one or more of a reactant’s covalent bonds and/or several noncovalent attractive interactions involving reactants are disrupted or completely broken. This process requires energy. At some point in the progress of the reaction, the energy reaches a maximum value. This state is a temporary, short-lived configuration of atoms called the transition state. In chapter 6 you learned that amount of energy required to reach the transition state is called the activation energy. As the reaction progresses, new covalent bonds and/or noncovalent attractive interactions form and generate the product; these processes release energy. The formation of the new covalent bonds and/or noncovalent attractive interactions is responsible for the decrease in energy that is seen in the diagram as the transition state changes to product. How does an enzyme increase the rate of a reaction? When substrates bind to an enzyme’s active site, interactions with the enzyme change the shape of the substrates (and enzyme) to a configuration that lowers the energy of the transition state (relative to an un-catalyzed reaction). Understanding Check: Determine whether each of the following statements are true or false. a. A catalyzed reaction has a lower activation energy than an un-catalyzed reaction. b. The greater the activation energy, the faster the reaction rate. c. At equilibrium, a catalyzed reaction will result in a greater amount of products than would an un-catalyzed reaction. 35 Almost all enzyme names use the “_________” suffix. Enzymes are named and categorized based on their __________________ and/or the ______________ that they catalyze. The table below lists some of the classes of enzymes, the reactions they catalyze, and some examples. Cofactors A ___________________ is defined as a non protein compound that must be permanently or temporarily bound to an enzyme in order for the enzyme to function. • Example: A nickel ion (Ni2+) must be bound to a urease enzyme in order for the enzyme to catalyze the conversion of urea to ammonium and bicarbonate. Cofactors are either inorganic ions or organic compounds. • When cofactors are organic compounds, they are often referred to as ____________________. In most cases, coenzymes are actually one of the __________________ in the catalyzed reaction. • The reason that certain substrates are also referred to as coenzymes is that they are common substrates to many different enzymatic reactions in which they the donate atoms or groups of atoms to other substrates or accept atoms or groups of atoms from other substrates. • For example, ATP and ADP are classified as coenzymes because they are involved in the transfer of phosphoryl groups (-PO3-) in many different enzymatically catalyzed reactions. • Many coenzymes are derived from dietary ______________. Understanding Check: Enzyme Specificity Choose one of the enzyme classes (from the table above) that would catalyze each of the following reactions. a. The conversion of a cis double bond to a trans double bond. b. The digestion of fat. c. The conversion of starch to D-glucose. d. The conversion of a dipeptide into two amino acids. e. The hydrolysis of ATP to form ADP and Pi. 36 Some of the atom/group-transfer substrates that are also classified as coenzymes, and their dietary sources are listed below. Effect of Temperature on the Rates of Enzymatically Catalyzed Reactions A typical graph of the rate of an enzymatically catalyzed reaction vs. temperature is shown below. The temperature at which the rate of the reaction is greatest is called the enzyme’s __________________ temperature. The reason that the reaction rate does not continue to increase after reaching the optimum temperature is that the enzyme begins to denature at the higher temperature. An enzyme’s optimum temperature is usually very close to the normal temperature of the organism in which it exists. • For example, the optimum temperature of most human enzymes is at normal body temperature (~37 oC), as depicted in the graph. Effect of pH on the Rates of Enzymatically Catalyzed Reactions A typical graph of the rate of an enzymatically catalyzed reaction vs. pH is shown below. The pH at which the rate of an enzymatically catalyzed reaction is greatest is called the enzyme’s _______________ pH. An enzyme’s optimum pH is usually very close to the normal pH of the region of an organism in which the enzyme exists. • For example, the normal pH in most regions of the body is about 7.4 (physiological pH), so the optimum pH for enzymes found in these regions is also near 7.4 (as depicted in the graph). As the pH increases or decreases from its optimum pH value, the reaction rate decreases because of enzyme denaturation. Not all parts of the body have a normal pH near 7.4. The stomach has a normal pH range of 1 to 3. It is not surprising that the digestive enzyme called pepsin, which functions in the stomach, has an optimum pH of 2. 37 Control of Enzymatic Reactions All life forms employ reaction regulation mechanisms that involve controlling enzymatically-catalyzed reactions by processes called enzyme ___________________ and enzyme ___________________. The body uses chemical feedback systems that can increase or decrease an enzyme's ability to catalyze a reaction. The amount of substrate that an enzyme converts to product (per second) is referred to as the enzyme’s “__________________.” 1) Enzyme Inhibition When a particular molecule (or ion) forms a covalent or noncovalent bond with an enzyme, it can result in a decrease in the enzyme’s activity. A species that decreases a particular enzyme’s activity is called an ______________________. • • Unlike temperature, pH, and denaturing agents, which affect all types of enzymes, inhibitors will only affect specific enzymes. Enzyme inhibition can be classified as __________________ inhibition or _______________ inhibition. i) Irreversible inhibition Irreversible inhibition occurs when an inhibitor reacts with an enzyme, forming a new and ___________ covalent bond to the enzyme. • In almost all cases of irreversible inhibition, the new bond is made to the enzyme’s active site, which results in complete and permanent loss of the enzyme’s activity. • In order to re-initiate catalysis, an organism must produce new enzymes (in the absence of the inhibitor). An example of irreversible inhibition is aspirin’s mode of operation. Aspirin irreversibly inhibits the cyclooxygenase (COX) enzyme, which catalyzes one of the reactions involved in prostaglandin production. Prostaglandins have a wide range of biological effects, including causing pain, inflammation, and fever. In order to prevent pain, inflammation, and fever, we use aspirin (or other nonsteroidal anti inflammatory drugs (NSAIDs)). Irreversible inhibition occurs when aspirin reacts with an amino acid side-chain in the COX enzyme's active site, as illustrated below. In this reaction, an acetyl group from aspirin is exchanged for a hydrogen atom (H) from a particular side-chain in the COX enzyme's active site. When an acetyl group is bonded to the enzyme’s active site, it is no longer possible for substrates to bind to the enzyme, and therefore the enzyme is permanently inactivated. 40 The regulation of a metabolic pathway by the inhibition of an enzyme is called ________________ inhibition. An example of feedback inhibition is the regulation of glycolysis. Glycolysis is a metabolic pathway that involves a series of ten chemical reactions. It is used by organisms to convert glucose to ATP, NADH, and pyruvate. If the concentrations of these products are sufficient, it would be inefficient and potentially harmful to continue to produce them. Each of the ten reactions in the glycolysis pathway requires an enzyme. Three of the enzymes in the pathway are regulated by inhibitors in the feedback inhibition process. 2) Enzyme Activation Enzyme activation can occur when an “activator” binds to an enzyme and _______________ its activity. Binding of the activator species to an enzyme induces changes in the active site that increases the enzyme's activity. Just like the substrates and enzyme inhibitors, enzyme activators are specific for a particular enzyme or group of enzymes. Enzyme activators can regulate metabolic pathways by activating one or more of the pathway’s enzymes. • Increasing the production of a metabolic pathway by an enzyme activator is called ______________ feedback. • An example of positive feedback is the activation of pyruvate kinase, an enzyme used in one of the glycolysis reactions. Pyruvate kinase is activated by PEP, which is also one of its own substrates (PEP). One last note on the control of enzyme activity: In this section, I discussed how enzyme activity could be decreased or increased by the binding of inhibitors or activators (respectively). Nature employs additional strategies in order to increase or decrease enzyme activity. In some cases, one enzyme catalyzes the breaking of chemical bonds, or the formation of new bonds, in a second enzyme in order to activate or deactivate the second enzyme. The details of these processes are beyond the scope of this course, however, you should know that this type of activation and deactivation is commonly employed by organisms to regulate metabolic pathways. Examples of the Involvement of Enzymes in Disease The underproduction or overproduction of enzymes, or the inability of an organism to control enzymes can lead to ______________. When such diseases result from a defect (mutation) in a gene that is responsible for the production of a particular enzyme, they are categorized as genetic diseases. In the next chapter, you will learn details of how the information in DNA is used to produce proteins (including enzymes). There are thousands of different types of enzymes that are produced in the body, and the inability to correctly produce or control just one type of enzyme could result in death. You may recall that I discussed Tay-sach’s and other sphingolipidosis genetic diseases that result from a deficiency of the enzymes responsible for the breakdown of sphingolipids. Although some forms of sphingolipidosis are treatable with enzyme replacement therapy, most sphingolipidosis cases result in death before five years of age. Enzyme replacement therapy is also used to treat other enzyme deficiencies, such as lactose intolerance (deficient lactase enzyme activity) and exocrine pancreatic deficiency (insufficient pancreatic production of digestive enzymes). 41 Another example of disease that is related to enzymes occurs when DNA is replicated. Before a cell divides, a duplicate copy of its DNA must be made. The new DNA is “proofread” for errors and then repaired. DNA repair enzymes catalyze the repair of mistakes made in the DNA replication process. If an individual’s DNA repair enzymes are not functional, this results in an accumulation of new mutations, and leads to various types cancer. Phenylketonuria (PKU) is an enzyme-related disease that can be controlled by a special diet. PKU is caused by deficient activity of the phenylalanine hydroxylase enzyme (PAH). This enzyme is responsible for breaking down excess phenylalanine (an amino acid). When the PAH enzyme is not fully functional, high levels of phenylalanine result, which affects brain development and causes intellectual disabilities, seizures, and other medical issues. If PKU is diagnosed and treated early, the damaging effects can be minimized and normal mental development can occur. For individuals with PKU, the consumption of foods rich in phenylalanine residues, such as meats and nuts, can be poisonous. Treatment of PKU is a strict life-time diet that restricts phenylalanine-containing foods, and includes dietary supplementation of the non phenylalanine amino acids, and other nutrients. Individuals with PKU must be careful not to consume the artificial sweetener called aspartame (NutraSweet) because phenylalanine is produced when aspartame is broken down in the body.