SAS for Biochemistry (BIO 024) Module #4.pdf

Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________...

Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ Lesson title: ENZYMES Materials: Lesson Objectives: by the end of this module, you Pen, SAS should be able to 1. Define common terminologies under enzyme References: 2. Determine the different classifications of enzyme ▪ stoker, H. S. (2017).Biochemistry (3rd and types of specificities ed.). (M. Finch, Ed.) Belmont CA, USA 3. Interpret the illustration and graphs depicting the ▪ Ferrier, D. (2017). Lippincott's Illustrated enzyme activity, factors affecting the enzyme Biochemistry (7 ed.). Lippincott Williams & Wilkins,. activity and enzyme inhibition ▪ Nemire, Ruth & Kier, Karen. (2009) 4. Provide examples of the medical uses of Pharmacy Student Survival Guide, 2nd enzymes. ed. Mc. Graw Hill 5. Productivity Tip: Try doing a house tour before starting this module. Take a quick look at some important things that makes your daily activities moves faster. This is to give your brain an idea of what’s coming - it’s like watching a trailer of a movie. Doing this for a minute will help your brain organize your thoughts before studying. A. LESSON PREVIEW/REVIEW 1) Introduction (1 min) Virtually all reactions in the body are mediated by enzymes (Greek word en “in” and zyme “yeast”), which are protein catalysts that increase the rate of reactions without being changed in the overall process. Among the many biologic reactions that are energetically possible, enzymes selectively channel reactants (called substrates) into useful pathways. Enzymes thus direct all metabolic events. This module examines the nature of these catalytic molecules, their mechanism of action and their importance. (Stoker, 3rd ed.) This document is the property of PHINMA EDUCATION Page |1  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ 2) Activity 1: What I Know Chart, part 1 (3 mins) Instructions: "In this chart, reflect on what you know now. Do not worry if you are sure or not sure of your answers. This activity simply serves to get you started on thinking about our topic. Answer only the first column, "What I know" based on the question of the second column. Leave the third column "What I learned" blank at this time. What I Know Questions: What I Learned (Activity 4) 1. What affects enzyme activity, pH, temperature or concentrations? 2. Is it correct to say that, drugs are enzyme inhibitors? Support your answer. 3. Enzymes are also biochemically used for diagnostic purposes, True or False? B.MAIN LESSON A. Activity 2: Content notes (60 min). Instructions: Please make your own mind map or outline. Note, it will help if you check the skill building activity first. However, do not answer the activity side by side with your notes so that you can assess your learning later better. I.NOMENCLATURE ------------------------------------------------------------------------------------- Each enzyme is assigned two names. The first is its short, recommended name, convenient for everyday use. The second is the more complete systematic name, which is used when an enzyme must be identified without ambiguity. A. Recommended name Most commonly used enzyme names have the suffix “-ase” attached to the substrate of the reaction (for example, glucosidase and urease), or to a description of the action performed (for example, lactate dehydrogenase and adenylyl cyclase). [Note: Some enzymes retain their original trivial names, which give no hint of the associated enzymic reaction, for example, trypsin and pepsin.] B. Systematic name In the systematic naming system, enzymes are divided into six major classes (Figure 2.1), each with numerous subgroups. For a given enzyme, the suffix -ase is attached to a fairly complete description of the chemical reaction catalyzed, including the names of all the substrates; for example, lactate: NAD+ oxidoreductase. [Note: Each enzyme is also assigned a classification number.] The systematic names are unambiguous and informative, but are frequently too cumbersome to be of general use. (Lippincott, 7th ed.) This document is the property of PHINMA EDUCATION Page |2  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ (Stoker, 3rd ed.) This document is the property of PHINMA EDUCATION Page |3  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ CLASSIFICATION OF ENZYME BASED ON THE REACTION BEING CATALYZED Main Class Selected Type of Reaction Catalyzed subclasses oxidases oxidation of a substrate OXIDOREDUCTAS reductases reduction of a substrate ES dehydrogenases introduction of double bond (oxidation) by formal removal of two H atoms from substrate, the H being accepted by a coenzyme Transaminases transfer of an amino group between substrates TRANSFERASES Kinases transfer of a phosphate group between substrates Lipases hydrolysis of ester linkages in lipids Proteases hydrolysis of amide linkages in proteins HYDROLASES Nucleases hydrolysis of sugar–phosphate ester bonds in nucleic acids Carbohydrases hydrolysis of glycosidic bonds in carbohydrates phosphatases hydrolysis of phosphate–ester bonds dehydratases removal of H2O from a substrate decarboxylases removal of CO2 from a substrate LYASES deaminases removal of NH3 from a substrate hydratases addition of H2O to a substrate Racemases conversion of D isomer to L isomer,or vice versa ISOMERASES Mutases transfer of a functional group from one position to another in the same molecule Synthetases formation of new bond between two substrates, with participation of ATP LIGASES Carboxylases formation of new bond bet. a substrate and CO2, with participation of ATP II.PROPERTIES OF ENZYME (Lippincott, 7th ed) ----------------------------------------------------------------------------------------------------------------------------------------- Enzymes are protein catalysts that increase the velocity of a chemical reaction, and are NOT consumed during the reaction. [Note: Some RNAs can act like enzymes, usually catalyzing the cleavage and synthesis of phosphodiester bonds. RNAs with catalytic activity are called ribozymes, and are much less commonly encountered than protein catalysts.] A. Active site Enzyme molecules contain a special pocket or cleft called the active site. The active site (10- 20% of the entire protein structure) contains amino acid side chains that participate in substrate binding and catalysis (Figure 2.2). The substrate binds the enzyme, forming an enzyme–substrate (ES) complex. Binding is thought to cause a conformational change in the enzyme (induced fit) that allows catalysis. ES is converted to an enzyme–product (EP) complex that subsequently dissociates to enzyme and product. 2.2 Substrate - is the reactant in an enzyme-catalysed reaction This document is the property of PHINMA EDUCATION Page |4  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ ▪ the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. ▪ The active site consists of residues that form temporary bonds with the substrate (binding site) and residues that catalyse a reaction of that substrate (catalytic site). Enzyme–substrate complex is the intermediate reaction species that is formed when a substrate binds to the active site of an enzyme. 2 types of ESC or Models of enzyme activity 1. Lock and key model - the active site in the enzyme has a fixed, rigid geometrical conformation. Only substrates with a complementary geometry can be accommodated at such a site, much as a lock accepts only certain keys. This is the simplest type and a product of enzyme specificity. 2. Induced fit model- allows for small changes in the shape or geometry of the active site of an enzyme to accommodate a substrate. The induced fit is a result of the enzyme’s flexibility; it adapts to accept the incoming substrate. 2.3 B. Catalytic efficiency Enzyme-catalyzed reactions are highly efficient, proceeding from 103–108 times faster than uncatalyzed reactions. The number of molecules of substrate converted to product per enzyme molecule per second is called the turnover number, or kcat and typically is 102–104s-1. C. Specificity Enzymes are highly specific, interacting with one or a few substrates and catalyzing only one type of chemical reaction. [Note: The set of enzymes made in a cell determines which metabolic pathways occur in that cell.] This document is the property of PHINMA EDUCATION Page |5  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ 4 TYPES OF SPECIFICITY ABSOLUTE GROUP enzyme will catalyze only one reaction. the enzyme will act only on molecules that Ex. Catalase enzyme catalyzes only have a specific functional group, such as Hydrogen peroxide (H2O2) to H2O and hydroxyl, amino, or phosphate groups. O2 Ex. Carboxypeptidase is group-specific; it cleaves amino acids, one at a time, from the carboxyl end of a peptide chain. LINKAGE STEREOCHEMICAL the enzyme will act on a particular type the enzyme will act on a particular of chemical bond, irrespective of the rest stereoisomer. Chirality is inherent in an enzyme of the molecular structure. active site because amino acids are chiral Ex. Phosphatases compounds. An L-amino acid oxidase will hydrolyze phosphate-ester bonds in all catalyze the oxidation of the L-form of an amino types of phosphate esters acid but not the D-form of the same amino acid D. Structural class, Holoenzymes, apoenzymes, cofactors, and coenzymes Enzymes can be divided into two general structural classes: simple enzymes and conjugated enzymes. A simple enzyme is an enzyme composed only of protein (amino acid chains). A conjugated enzyme is an enzyme that has a nonprotein part in addition to a protein part. By itself, neither the protein part nor the nonprotein portion of a conjugated enzyme has catalytic properties. Some enzymes require molecules other than proteins for enzymic activity. The term holoenzyme refers to the active enzyme with its nonprotein component, whereas the enzyme without its nonprotein moiety or the protein part of the conjugated enzyme is termed an apoenzyme and is inactive. If the nonprotein moiety is a metal ion such as Zn2+ or Fe2+, it is called a cofactor. If it is a small organic molecule, it is termed a coenzyme. Coenzymes that only transiently associate with the enzyme are called cosubstrates. Cosubstrates dissociate from the enzyme in an altered state (NAD+ is an example). If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group (FAD is an example). Coenzymes frequently are derived from vitamins (see vitamin module). For example, NAD+ contains niacin and FAD contains riboflavin. Apoenzyme + nonprotein moiety (cofactor) = holoenzyme E. Regulation Enzyme activity can be regulated, that is, increased or decreased, so that the rate of product formation responds to cellular need. F. Location within the cell Many enzymes are localized in specific organelles within the cell (Figure 2.3). Such compartmentalization serves to isolate the reaction substrate or product from other competing reactions. This provides a favorable environment for the reaction, and organizes the thousands of enzymes present in the cell into purposeful pathways. This document is the property of PHINMA EDUCATION Page |6  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ III.HOW ENZYMES WORK (MECHANISM OF ACTION) (Lippincott, 7th ed) ------------------------------------------------------------------------------------------------------------------------------ Two different perspectives: First, catalysis in terms of energy changes that occur during the reaction, that is, enzymes provide an alternate, energetically favorable reaction pathway different from the uncatalyzed reaction. Second, describes how the active site chemically facilitates catalysis. A. Energy changes occurring during the reaction Virtually all chemical reactions have an energy barrier separating the reactants and the products. This barrier, called the free energy of activation, is the energy difference between that of the reactants and a high-energy intermediate that occurs during the formation of product. For example, Figure 2.4 shows the changes in energy during the conversion of a molecule of reactant A to product B as it proceeds through the transition state (high-energy intermediate), T*: A↔T*↔B 1.Free energy of activation: The peak of energy in Figure 2.4 is the difference in free energy between the reactant and T*, where the high- energy intermediate is formed during the conversion of reactant to product. Because of the high (ΔG) free energy of activation, the rates of uncatalyzed chemical reactions are often slow. 2.4 2.Rate of reaction: For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state. In the absence of an enzyme, only a small proportion of a population of molecules may possess enough energy to achieve the transition state between reactant and product. The rate of reaction is determined by the number of such energized molecules. In general, the lower the free energy of activation, the more molecules have sufficient energy to pass through the transition state, and, thus, the faster the rate of the reaction. 3.Alternate reaction pathway: An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower free energy of activation (Figure 2.4). The enzyme does not change the free energies of the reactants or products and, therefore, does not change the equilibrium of the reaction (see illustration). It does, however, accelerate the rate with which equilibrium is reached. This document is the property of PHINMA EDUCATION Page |7  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ B.Chemistry of the active site The active site is not a passive receptacle for binding the substrate, but rather is a complex molecular machine employing a diversity of chemical mechanisms to facilitate the conversion of substrate to product. A number of factors are responsible for the catalytic efficiency of enzymes, including the following: 1. Transition-state stabilization: The active site often acts as a flexible molecular template that binds the substrate and initiates its conversion to the transition state, a structure in which the bonds are not like those in the substrate or the product (see T* at the top of the curve in Figure 2.4). By stabilizing the transition state, the enzyme greatly increases the concentration of the reactive intermediate that can be converted to product and, thus, accelerates the reaction. 2. 2. Catalysis: The active site can provide catalytic groups that enhance the probability that the transition state is formed. In some enzymes, these groups can participate in general acid-base catalysis in which amino acid residues provide or accept protons. In other enzymes, catalysis may involve the transient formation of a covalent ES complex. [Note: Chymotrypsin , an enzyme of protein digestion in the intestine, includes general base, general acid, and covalent catalysis] 1. 3. Visualization of the transition state: The enzyme-catalyzed conversion of substrate to product can be visualized as being similar to removing a sweater from an uncooperative infant (see figure at the side). The process has a high energy of activation because the only reasonable strategy for removing the garment (short of ripping it off) requires that the random flailing (wave or swing or cause to wave or swing wildly) of the baby results in both arms being fully extended over the head—an unlikely posture. However, we can envision a parent acting as an enzyme, first coming in contact with the baby (forming ES), then guiding the baby’s arms into an extended, vertical position, analogous to the ES transition state. This posture (conformation) of the baby facilitates the removal of the sweater, forming the disrobed baby, which here represents product. [Note: The substrate bound to the enzyme (ES) is at a slightly lower energy than unbound substrate (S) and explains the small “dip” in the curve at ES.] This document is the property of PHINMA EDUCATION Page |8  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ IV. FACTORS AFFECTING ENZYMATIC ACTIVITY (Lippincott, 7th ed.) ------------------------------------------------------------------------------------------------ This section describes factors that influence the reaction velocity of enzymes. Enzymic responses to these factors give us valuable clues as to how enzymes function in living cells (that is, in vivo). A. Substrate concentration 1. Maximal velocity: The rate or velocity of a reaction (v) is the number of substrate molecules converted to product per unit time; velocity is usually expressed as μmol of product formed per minute. The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) Figure 2.5: effect of substrate is reached (Figure 2.5). The leveling off of the reaction rate at concentration on rxn velocity high substrate concentrations reflects the saturation with substrate of all available binding sites on the enzyme molecules present. Saturation means that all enzyme active sites were fully occupied and so the reaction rate remains constant 2. Hyperbolic shape of the enzyme kinetics curve: Most enzymes show Michaelis-Menten kinetics (see next pages), in which the plot of initial reaction velocity (vo) against substrate concentration ([S]), is hyperbolic (similar in shape to that of the oxygen-dissociation curve of myoglobin, see illustration). In contrast, allosteric enzymes do not follow Michaelis-Menten kinetics and show a sigmoidal curve (see illustation) that is similar in shape to the oxygen dissociation curve of hemoglobin (see illustration). B. Temperature 1. Increase of velocity with temperature: The reaction velocity increases with temperature until a peak/maximum velocity is reached (Figure 2.6). This increase is the result of the increased number of molecules having sufficient energy to pass over the energy barrier and form the products of the reaction. Note: Optimum temperature is the temperature at which an enzyme exhibits maximum activity (that is substrate is Figure 2.6: effect of temperature converted to product). on an enzyme reaction This document is the property of PHINMA EDUCATION Page |9  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ 2. Decrease of velocity with higher temperature: Further elevation of the temperature results in a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme (see Figure 2.6). The optimum temperature for most human enzymes is between 35 and 40°C. Human enzymes start to denature at temperatures above 40°C, but thermophilic bacteria found in the hot springs have optimum temperatures of 70°C. C. pH 1. Effect of pH on the ionization of the active site: The concentration of H+ affects reaction velocity in several Figure 2.7: effect of pH on an enzyme ways. First, the catalytic process usually requires that the reaction enzyme and substrate have specific chemical groups in either an ionized or un-ionized state in order to interact. For example, catalytic activity may require that an amino group of the enzyme be in the protonated form (–NH3+). At alkaline pH, this group is deprotonated, and the rate of the reaction, therefore, declines. 2. Effect of pH on enzyme denaturation: Extremes of pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid side chains. 3. The pH optimum varies for different enzymes: The pH at which maximal enzyme activity is achieved is different for different enzymes, and often reflects optimum pH. For example, pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other enzymes, designed to work at neutral pH, are denatured by such an acidic environment (Figure 2.7). (You may check for extremophiles or enzymes that can live in more harsh environment.) D. Enzyme concentration Because enzymes are not consumed in the reactions they catalyze, the cell usually keeps the number of enzymes low compared with the number of substrate molecules. This is efficient; the cell avoids paying the energy costs of synthesizing and maintaining a large work force of enzyme molecules. Thus, in general, the concentration of substrate in a reaction is much higher than that of the enzyme. If the amount of substrate present is kept constant and the enzyme concentration is increased, the reaction rate increases because more substrate molecules can be accommodated in a given amount of time (see Figure 2.). The greater the enzyme concentration, the greater the reaction rate. Figure 2.8: effect of eznyme concentration on rxn velocity This document is the property of PHINMA EDUCATION P a g e | 10  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ V. MICHAELIS-MENTEN EQUATION (Don’t worry there’ll be no computation yet) --------------------------------------------------------------------------------------------------------------------------------------- A. Reaction model Leonor Michaelis and Maude Menten proposed a simple model that accounts for most of the features of enzyme-catalyzed reactions. In this model, the enzyme reversibly combines with its substrate to form an ES complex that subsequently yields product, regenerating the free enzyme. The model, involving one substrate molecule, is represented below: where S is the substrate E is the enzyme ES is the enzyme–substrate complex P is the product k1, k-1, and k2 are rate constants B.Michaelis-Menten equation The Michaelis-Menten equation describes how reaction velocity varies with substrate concentration: The following assumptions are made in deriving the Michaelis- Menten rate equation: 1. Relative concentrations of E and S: The concentration of substrate ([S]) is much greater than the concentration of enzyme ([E]), so that the percentage of total substrate bound by the enzyme at any one time is small. 2. Steady-state assumption: 2.9 [ES] does not change with time (the steady-state assumption), that is, the rate of formation of ES is equal to that of the breakdown of ES (to E + S and to E + P). In general, an intermediate in a series of reactions is said to be in steadystate when its rate of synthesis is equal to its rate of degradation. This document is the property of PHINMA EDUCATION P a g e | 11  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ 3. Initial velocity: Initial reaction velocities (vo) are used in the analysis of enzyme reactions. This means that the rate of the reaction is measured as soon as enzyme and substrate are mixed. At that time, the concentration of product is very small and, therefore, the rate of the back reaction from P to S can be ignored. C.Important conclusions about Michaelis-Menten kinetics 1. Characteristics of Km: Km—the Michaelis constant—is characteristic of an enzyme and its particular substrate, and reflects the affinity of the enzyme for that substrate. Km is numerically equal to the substrate concentration at which the reaction velocity is equal to 1⁄2Vmax. Km does not vary with the concentration of enzyme. a. Small Km: A numerically small (low) Km reflects a high affinity of the enzyme for substrate, because a low concentration of substrate is needed to half-saturate the enzyme—that is, to reach a velocity that is 1⁄2Vmax (Figure 2.9). b. Large Km: A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration of substrate is needed to half-saturate the enzyme. 2. Relationship of velocity to enzyme concentration: The rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations. For example, if the enzyme concentration is halved, the initial rate of the reaction (vo), as well as that of Vmax, are 2.10 reduced to half that of the original. 3. Order of reaction: When [S] is much less than Km, the velocity of the reaction is approximately proportional to the substrate concentration (Figure 2.10). The rate of reaction is then said to be first order with respect to substrate. When [S] is much greater than Km, the velocity is constant and equal to Vmax. The rate of reaction is then independent of substrate concentration, and is said to be zero order with respect to substrate concentration (see Figure 2.10). D. Lineweaver-Burk plot 2.11 When vo is plotted against [S], it is not always possible to determine when Vmax has been achieved, because of the gradual upward slope of the hyperbolic curve at high substrate concentrations. However, if 1/vo is plotted versus 1/[S], a straight line is obtained (Figure 2.11). This plot, the Lineweaver-Burk plot (also called a double-reciprocal plot) can be used to calculate Km and Vmax, as well as to determine the mechanism of action of enzyme inhibitors. 1. The equation describing the Lineweaver-Burk plot is: This document is the property of PHINMA EDUCATION P a g e | 12  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ VI. INHIBITION OF ENZYME ACTIVITY --------------------------------------------------------------------------------------------------------------------------------------- Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called an inhibitor. In general, irreversible inhibitors bind to enzymes through covalent bonds. Reversible inhibitors typically bind to enzymes through noncovalent bonds, thus dilution of the enzyme–inhibitor complex results in dissociation of the reversibly bound inhibitor, and recovery of enzyme activity. The two most commonly encountered types of reversible inhibition are competitive and noncompetitive. A. Competitive inhibition This type of inhibition occurs when the inhibitor binds reversibly to the same site that the substrate would normally occupy and, therefore, competes with the substrate for that site. 1. Effect on Vmax: The effect of a competitive inhibitor is reversed by increasing [S]. At a sufficiently high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of inhibitor (Figure 2.12). 2. Effect on Km: A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve 1⁄2Vmax. 3. Effect on the Lineweaver-Burk plot: Competitive inhibition shows a characteristic Lineweaver- Burk plot in which the plots of the inhibited and uninhibited reactions intersect on the y-axis at 1/Vmax (Vmax is unchanged). The inhibited and uninhibited reactions show different x-axis intercepts, indicating that the apparent Km is increased in the presence of the competitive inhibitor because -1/Km moves closer to zero from a negative value (see Figure 2.12). 2.12 This document is the property of PHINMA EDUCATION P a g e | 13  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ 4.Statin drugs as examples of competitive inhibitors: This group of antihyperlipidemic agents competitively inhibits the first committed step in cholesterol synthesis. This reaction is catalyzed by hydroxymethylglutaryl– CoA reductase ( HMG-CoA reductase). Statin drugs, such as atorvastatin (Lipitor) and pravastatin (Pravachol),1 are structural analogs of the natural substrate for this enzyme, and compete effectively to inhibit HMG-CoA reductase. By doing so, they inhibit de novo cholesterol synthesis, thereby lowering plasma cholesterol levels (Figure 2.13). B. Noncompetitive inhibition This type of inhibition is recognized by its characteristic effect on Vmax (Figure 2.14). Noncompetitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme. The noncompetitive inhibitor can bind either free enzyme or the ES complex, thereby preventing the reaction from occurring (Figure 2.15). 1. Effect on Vmax: Noncompetitive inhibition cannot be overcome 2.13 by increasing the concentration of substrate. Thus, noncompetitive inhibitors decrease the apparent Vmax of the reaction. 2. Effect on Km: Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme. Thus, the enzyme shows the same Km in the presence or absence of the noncompetitive inhibitor. Km is unchanged. 3. Effect on Lineweaver-Burk plot: Noncompetitive inhibition is readily differentiated from competitive inhibition by plotting 1/vo versus 1/[S] and noting that the apparent Vmax decreases in the presence of a noncompetitive inhibitor, whereas Km is unchanged (see Figure 2.14). 2.14 2.15 This document is the property of PHINMA EDUCATION P a g e | 14  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ C. IRREVERSIBLE INHIBITION Irreversible Inhibition An irreversible enzyme inhibitor is a molecule that inactivates enzymes by forming a strong covalent bond to an amino acid side-chain group at the enzyme’s active site. In general, such inhibitors do not have structures similar to that of the enzyme’s normal substrate. The inhibitor–active site bond is sufficiently strong that addition of excess substrate does not reverse the inhibition process. Thus the enzyme is permanently deactivated. The actions of chemical warfare agents (nerve gases) and organophosphate insecticides are based on irreversible inhibition. D. Enzyme inhibitors as drugs At least half of the ten most commonly dispensed drugs in the United States act as enzyme inhibitors. For example, the widely prescribed β-lactam antibiotics, such as penicillin and amoxicillin, act by inhibiting enzymes involved in bacterial cell wall synthesis. Drugs may also act by inhibiting extracellular reactions. This is illustrated by angiotensin-converting enzyme (ACE) inhibitors. They lower blood pressure by blocking the enzyme that cleaves angiotensin I to form the potent vasoconstrictor, angiotensin II. These drugs, which include captopril, enalapril, and lisinopril, cause vasodilation and a resultant reduction in blood pressure. Aspirin, a non-prescription drug irreversibly inhibits prostaglandins and thromboxane synthesis by inhibiting cyclooxygenase. VII. ENZYME REGULATION -------------------------------------------------------------------------------------------- A. Allosteric enzymes Are regulated by molecules called effectors (also called modifiers) that bind noncovalently at a site other than the active site. These enzymes are usually composed of multiple subunits, and the regulatory (allosteric) site that binds the effector may be located on a subunit that is not itself catalytic. The presence of an allosteric effector can alter the affinity of the enzyme for its substrate, or modify the maximal catalytic activity of the enzyme, or both. Effectors that inhibit enzyme activity are termed negative effectors, whereas those that increase enzyme activity are called positive effectors. Positive and negative effectors can affect the affinity of the enzyme for its substrate (K0.5), modify the maximal catalytic activity of the enzyme (Vmax), or both (Fig. 2.16). Allosteric enzymes frequently catalyze the committed step, often the rate limiting step, early in a pathway. 2.16 This document is the property of PHINMA EDUCATION P a g e | 15  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ B. Regulation of enzymes by covalent modification Many enzymes may be regulated by covalent modification, most frequently by the addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. 1. Phosphorylation and dephosphorylation: Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that use adenosine triphosphate (ATP) as a phosphate donor. Phosphate groups are cleaved from phosphorylated enzymes by the action of phosphoprotein phosphatases (Figure 2.17). 2. Response of enzyme to phosphorylation: Depending on the specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme. For example, 2.17 phosphorylation of glycogen phosphorylase (an enzyme that degrades glycogen) increases activity, whereas the addition of phosphate to glycogen synthase (an enzyme that synthesizes glycogen) decreases activity. C. Induction and repression of enzyme synthesis Cells can also regulate the amount of enzyme present by altering the rate of enzyme degradation or, more typically, the rate of enzyme synthesis. The increase (induction) or decrease (repression) of enzyme synthesis leads to an alteration in the total population of active sites. Enzymes subject to regulation of synthesis are often those that are needed at only one stage of development or under selected physiologic conditions. For example, elevated levels of insulin as a result of high blood glucose levels cause an increase in the synthesis of key enzymes involved in glucose metabolism. In contrast, enzymes that are in constant use are usually not regulated by altering the rate of enzyme synthesis. Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days), compared with allosterically or covalently regulated changes in enzyme activity, which occur in seconds to minutes. Figure 2.18 summarizes the common ways that enzyme activity is regulated. 2.18 This document is the property of PHINMA EDUCATION P a g e | 16  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ D. Proteolytic enzyme and zymogen : regulating cellular enzyme activity based on the production of enzymes in an inactive form. These inactive enzyme precursors are then “turned on” at the appropriate time. Such a mechanism for control is often encountered in the production of proteolytic enzymes. Because they would otherwise destroy the tissues that produce them, proteolytic enzymes are generated in an inactive form and then later, when they are needed, are converted to their active form. Digestive enzymes needs activator like HCl so that zymogen are converted to proteolytic enzyme. proteolytic enzyme : an enzyme that catalyzes the breaking of peptide bonds that maintain the primary structure of a protein. zymogen or proenzyme: is the inactive precursor of a proteolytic enzyme. EXAMPLES: ZYMOGEN PROTEOLYTIC ENZYME ▪ Pepsinogen ------- pepsin ▪ Tyrpsinogen ------- trypsin ▪ Chymotrypsinogen ------ chymotrypsin ▪ Angiotensinogen ------- angiotensin VIII. ENZYMES IN CLINICAL DIAGNOSIS --------------------------------------------------------------------------------------------------------------------------------- Plasma enzymes can be classified into two major groups. First, a relatively small group of enzymes are actively secreted into the blood by certain cell types. For example, the liver secretes zymogens (inactive precursors) of the enzymes involved in blood coagulation. Second, a large number of enzyme species are released from cells during normal cell turnover. These enzymes almost always function intracellularly, and have no physiologic use in the plasma. In healthy individuals, the levels of these enzymes are fairly constant, and represent a steady state in which the rate of release from damaged cells into the plasma is balanced by an equal rate of removal of the enzyme protein from the plasma. Increased plasma levels of these enzyme may indicate tissue damage (Figure 5.20). BIOCHEMICALLY IMPORTANT ENZYMES: (Nemire, 2nd ed) 1.CREATINE KINASE: is an enzyme that is found primarily in skeletal and cardiac muscle and in smaller fractions in the brain TYPES OF CK muscle (CK-MM), brain (CK-BB), cardiac tissue (CK-MB) - important marker in the diagnosis of acute myocardial infarction (AMI) This document is the property of PHINMA EDUCATION P a g e | 17  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ 2.CARDIAC TROPONIN Description Troponin I and T are sensitive markers of cardiac injury. Troponin I is found solely in the cardiac muscle, and Troponin T is found in both cardiac and skeletal muscle. Clinical Significance Troponin levels begin to rise within 4 hours of onset of chest pain. Levels should be drawn on admission and within 8 to 12 hours thereafter. Patients with elevated troponin levels are considered at high risk for a significant cardiac event. 3. GASTROINTESTINAL TESTS A.Alanine Aminotransferase/ serum glutamic pyruvic transaminase (SGPT). i. - liver tissue. It is also located in myocardial, muscle, and renal tissue ii. - considered a specific marker for liver disease B.Aspartate Aminotransferase/ serum glutamic oxaloacetic transaminase (SGOT i. - found in the liver. It is also present in the heart, kidney, pancreas, lungs, and skeletal muscle ii. For diagnosis of liver disease C.g-Glutamyl Transpeptidase o an enzyme found in the liver, kidney, and pancreas. GGT levels are useful in the diagnosis and monitoring of alcoholic liver disease o Increased GGT may be seen in alcoholic liver disease, metastatic liver disease, obstructive jaundice, cholelithiasis, and pancreatitis D.Lactate Dehydrogenase o enzyme involved in the interconversion of lactate and pyruvate. o is found in many tissues, including heart, brain, liver, skeletal muscle, kidneys, lungs, and RBCs. o LDH4 and LDH5 are present in liver tissue, and elevations may be seen in liver disease such as hepatitis and cirrhosis. o LDH1 and LDH2 may be useful in the diagnosis of myocardial infarction E. Lipase enzyme that aids in the digestion of fat. It is primarily secreted by the pancreas. useful in the diagnosis of pancreatitis and is considered a more specific marker for acute pancreatitis than amylase F.Amylase enzyme that aids in digestion by breaking down complex carbohydrates into simple sugars. The majority of amylase is produced in the pancreas and salivary glands, and lesser amounts are secreted by the fallopian tubes, lungs, thyroid, and tonsils Serum amylase levels are most often used in the diagnosis of acute pancreatitis This document is the property of PHINMA EDUCATION P a g e | 18  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ Condition Isoenzyme pattern Serum enzyme Major diagnostic use Muscular dystrophy Elevation of LDH1-3 Acid phosphate Prostate cancer Megaloblastic Large elevation of LDH1 Alkaline phosphatase Liver and bone disease anemia Sickle cell anemia Moderate elevation of LDH1 and Creatine phosphokinase Myocardial infarction LDH2 and muscle disorders Arthritis with joint Elevation of LDH5 Lactate dehydrogenase Myocardial infarction, infections leukemia, anemia Acute hepatitis Moderate elevation of LDH1, slight Renin Hypertension elevation of LDH2 Activity 3: Skill-building Activities (with answer key) (25 mins + 5 mins checking) A. Identification. Write the correct answer to the following before the number ____________1. The meaning of “zyme” in enzyme. ____________ 2. RNAs with catalytic activity are called _____ ____________3. is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. ____________ 4. is the intermediate reaction species that is formed when a substrate binds to the active site of an enzyme. ____________ 5. is the reactant in an enzyme-catalysed reaction B. Matching Type. Write the correct answer to the following before the number Column A1 Column B1 A. Absolute ________1. enzyme will catalyze only one reaction B. Group ________2. the enzyme will act only on molecules that C. Linkage have a specific functional group, such as hydroxyl, amino, D. Stereochemical or phosphate groups ________3. the enzyme will act on a particular stereoisomer ________4. the enzyme will act on a particular type of chemical bond, irrespective of the rest of the molecular structure This document is the property of PHINMA EDUCATION P a g e | 19  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ Column A2 Column B2 A. Oxidoreductases ________1. Oxidases B. Transferases C. Hydrolases ________2. Mutases D. Lyases E. Isomerases ________3. Decarboxylase F. Ligases ________4. Kinases ________5. Phosphatases ________6. Carboxylases Column A3 A. Substrate Concentration B. Temperature C. pH D. Enzyme concentration Column B3 ________1. _______ 3. ______2. ________ 4. This document is the property of PHINMA EDUCATION P a g e | 20  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ Column A4 A. Competitive inhibition B. Non-competitive inhibition C. Irreversible inhibition Column B4 ________1. \ ________ 2. Column A5 Column B5 A. CK – MM ________1. Creatinine kinase found in muscle B. CK – BB C. Troponin I ________2. Also known as alanine aminotransferase D. Troponin T E. SGPT ________3. found in both cardiac and skeletal muscle. F. SGOT ________4. Also known as aspartate aminotransferase ________5. Creatinine kinase found in brain This document is the property of PHINMA EDUCATION P a g e | 21  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ Activity 4: What I Know Chart, part 2 (2 mins) Instruction: To review what was learned from this session, please go back to Activity 1 and answer the “What I Learned” column. Notice and reflect on any changes in your answer Activity 5: Check for Understanding (20 mins) Instruction: Now it’s time for you to figure this one out on your own! Take time to read, analyze, and understand the following questions. For this instance, you will not have the chance to check if you have the correct answers since there are no more keys to correction. Write the letter of your choice. Good luck! 5. Inactive precursor of a proteolytic enzyme: For number 1 a. Holoenzyme c. Apoenzyme b. Zymogen d. Coenzyme 6. Enzymes, which are produced in inactive form in the living cells, are called a. Papain c. Lysozymes b. Apoenzymes d. Proenzymes 1. The enzyme-substrate complex and enzyme is represented by diagram a. A;C b.B;D c.C:B d.D:B e. C;A 7. Enzyme that is very useful in the diagnosis of infectious For numbers 2-4 hepatitis a. SGOT c. TPP b. SGPT d. NAD 8. The following are examples of a proteolytic enzyme, except? a. Angiotensin c. Pepsinogen b. Chymotrepsin d. Trypsin 9. Major diagnostic use of Creatine phosphokinase: 2. In the area marked A on the graph, many of the a. Hypertension D. Wilson’s disease enzyme molecules have active sites that are b. Prostate cancer a. Available for substrate binding c. Myocardial infarction and muscle disorders b. Occupied by the inhibitors c. Damaged by denaturants 10. This type of enzyme specificity states that some d. Saturated by the substrate enzymes are specific to only one isomer even if the e. Increasingly interacting with the substrate compound is one type of molecule a. Absolute specificity 3. What phenomenon explains the flat area between b. Group specificity points B and C? Still referring to the illustration. c. Stereochemical specificity a. The reaction has come to stop. d. Linkage specificity b. All of the enzyme’s active site are occupied c. The reaction has run out of substrate d. The enzyme has stopped working. 11. competitive inhibitor can e. the enzymes saturation curve is achieved. a. be nullified by increasing product concentration f. the reaction has dropped. b. be reversed by increasing substrate concentration c. speed up reaction decreasing the temperature 4. Which of these changes might increase the rate of d. be overcome by maintaining pH level the reaction beyond point C? Increase… a. Substrate concentration b. Enzyme concentration c. temperature d. water concentration e. nonspecific inhibitors This document is the property of PHINMA EDUCATION P a g e | 22  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ 12. It increases enzyme activity; the shape of the active site is changed such that it can more readily accept substrate. A. Competitive enzyme inhibitor B. Noncompetitive enzyme inhibitor C. Irreversible enzyme inhibitor D. Positive regulator 13. Fischer’s ‘lock and key’ model of the enzyme action implies that: a. The active site is complementary in shape to that of the substance only after interaction. b. The active site is complementary in shape to that of substance. c. Substrates change shape prior to active site interaction d. The active site is flexible and adjusts to substrate. 14. The following are examples of enzymes that exhibit group specificity, EXCEPT for a. Phosphatases c. Esterases b. Carboxypeptidase d. Glycosidases 1. Found solely on the cardiac muscles a. Troponin C b. Troponin T c. Troponin I d. Troponin I and T A. LESSON WRAP-UP 1) Activity 6: Thinking about Learning (5 mins) A. Work Tracker: You are done with this session! Let’s track your progress. Shade the session number you just completed. P1 P2 1 2 3 4 5 6 7 8 9 10 B. Think about your Learning: Tell me about your thoughts! Today’s topic is all about the enzymes. What interests you about the lesson today? What are the things that confuses you from the topic? ___________________________________________________________ ___________________________________________________________ ___________________________________________________________ ________________________________________________________ This document is the property of PHINMA EDUCATION P a g e | 23  Course Code: BIO 024 (Biochemistry/Biomolecules) Student Activity Sheet Module #4 Name: ____________________________________________________________ Class number: _______ Section: ____________ Schedule: ____________________________________ Date: _______________ KEY TO CORRECTIONS FOR ACTIVITY 3 A. Identification 1. Yeast 5. Substrate 2. Ribozyme 3. Active site 4. ESC or Enzyme - Substrate Complex B. Matching Type A1. A2. A3. A4. A5. 1. A 1. A 1. B 1. A 1. A 2. B 2. E 2. C 2. C 2. E 3. D 3. D 3. A 3. B 3. D 4. C 4. B 4. D 4. F 5. C 5. B 6. F SUGGESTED VIDEOS: Enzymes overview https://www.youtube.com/watch?v=qgVFkRn8f10 How enzymes work (energy of activation)? https://www.youtube.com/watch?v=wiIUS2LDCl8 https://www.youtube.com/watch?v=j00Ep0Byu0Y Factors affecting enzymatic activity: https://www.youtube.com/watch?v=dhAiTiqUv3w Michaelis-Menten Kinetics: Part 1(These are quite long or about 20min videos) https://www.youtube.com/watch?v=4eLjRcHnMCk Michaelis-Menten and Lineweaver-Burk Plot: Part 2 https://www.youtube.com/watch?v=y43pIHUtjeM Enzyme inhibition: (This is also quite long or about 20min videos) https://www.youtube.com/watch?v=jJUoQMLMV2E This document is the property of PHINMA EDUCATION P a g e | 24