UNIT Three- Enzymes (3).pptx PDF

UNIT THREE ENZYMES Lesson objectives  At the end of this section, students will be able to:  Define enzymes and activation energy  Explain how enzymes work  Describe the catalysis reaction of enzymes, activities and substrates  Nature of...

UNIT THREE ENZYMES Lesson objectives  At the end of this section, students will be able to:  Define enzymes and activation energy  Explain how enzymes work  Describe the catalysis reaction of enzymes, activities and substrates  Nature of enzymes  What are enzyme molecules like?  Enzymes can be defined more precisely as:  An enzymes is a globular proteins with a uniquely shaped active site.  Enzymes accelerate the rate of chemical reactions by lowering activation energy.  Activation energy is the minimum amount of energy required for the reactant to be converted to products.  Enzymes are biological catalyst for a specific reaction, but remain unaltered by the reaction.  All enzymes are globular proteins made up of chains of amino acids linked together by peptide bonds but not all biological catalysts are proteins.  Ribozymes: RNA molecules that exhibit catalytic activity in the absence of any protein component.  All globular proteins have unique tertiary structures which give them a unique shape.  A catalyst is a substance that speeds up a reaction; the reaction itself is unaltered. There is no overall change to:  the nature of the products  the energy change that takes place during the reaction  the catalyst itself Cont…  Enzymes are the mediators of metabolism.  All cells contain different enzymes.  Metabolism is the process of chemical and physical changes, including the breakdown (catabolism) and synthesis (anabolism) of molecules.  As catalysts, enzymes accelerate bond-breaking (catabolism), and bond forming (anabolism)processes to sustain life.  Enzymes do this by forming a complex with reactants, called an enzyme–substrate (ES) complex.  The distinguishing feature of an enzyme-catalyzed reaction is that it takes place within the confines of a pocket on the enzyme called the active site.  The structure of the active site accounts not only for the catalytic activity of the enzyme, but also for its specificity.  The molecule that is bound in the active site and acted upon by the enzyme is called the substrate, convert them into products of different molecules, and remain unchanged.  Cont…  The part of the enzyme molecule that is directly involved in binding the substrate is termed the active site.  The active site and the substrate(s) have complementary shapes.  Such binding requires less energy than uncatalyzed reactions.  The binding of substrate to enzyme is accomplished by the same types of noncovalent interactions (ionic bonds, hydrogen bonds, hydrophobic interactions) that determine the structure of the protein itself.  The active site contains amino acid side chain that participate in substrate binding and catalysis.  The substrate binds the enzyme, forming an enzyme– substrate (ES) complex.  Binding is thought to cause a conformational change in the active site of enzyme (induced fit) that allows catalysis.  ES is converted to an enzyme–product (EP) complex that subsequently dissociates to enzyme and product. Cont’d Fig.3.2 The hydrolysis of sucrose by sucrose Lab 3.1 Classroom experimental activities  Objectives: investigate the enzyme Salivary Amylase in mouth.  Sweet taste:  Did you ever wonder why rice, corn, and potatoes have a slightly sweet taste when you chew them?  This is because they contain large amounts of starch, a polymeric carbohydrate consisting of numerous glucose units joined by glycosidic bonds.  Starch itself is mostly tasteless, but when it is degraded the glucose molecules reach your taste buds and the magic happens.  The enzyme responsible for starch degradation is called amylase, and is found in saliva, among other places.  Ptyalin is the starch hydrolyzing enzyme secreted by salivary glands in human beings. It is also called as salivary amylase.  This process continues in the small intestine, where amylase produced by the pancreas performs the final steps of carbohydrate digestion.  In addition to amylase, our saliva also contains many other enzymes including lipases, lysozyme (antimicrobial). Cont…  The distinguishing feature of an enzyme- catalyzed reaction is that it takes place within the confines of a pocket on the enzyme called the active site.  The structure of the active site accounts not only for the catalytic activity of the enzyme, but also for its specificity.  Most enzymes are capable of binding only one or a small number of closely related biological molecules. Properties and Functions of Enzyme  Afterthe successful completion of this section, the student will be able to:  Identify the properties of enzymes  Explain the action of each property  Describe the functions properties  General Properties of an Enzyme  What are the enzyme properties?  The general properties of enzymes are the nature of both their physical and chemical properties.  A. The physical properties of enzymes Cont’d  Enzyme denaturation /unfolding of protein/:  is the process of breaking the intra and inter-molecular non-covalent bonds that distort the shape and active site of the enzymes, leading to the loss of enzyme activity.  The denatured protein changes in its physical state while maintaining the chemical composition.  Enzymes loses its native conformation or three-dimensional structure.  Enzymes are denatured by:  high heat above the organisms optimal temperature (above 40ºC),  change in the pH (too low or too high),  heavy metals ionization can disrupt the function of protein by forming complexes with functional side chains or oxidizing amino acid side chains.  high salt concentrations denature enzymes by stripping away the water molecules from the protein surface and breaking the chemical bonds that hold the protein structure together.  solvents and other reagents  Enzyme solubility:  Enzymes are soluble in a variety of solutions including water, salt (NaCl), diluted glycerol and alcohol causing denaturation.  The colloidal nature of enzyme:  the tendency of having little or no dialysis or pass cross the semipermeable membrane due to the large size or high molecular weight.  Molecular weights of enzymes:  Enzymes are large protein that contain polypeptide chains of various amino acid sequences.  The amino acids are held together by approximately 200 to 300 peptide linkages.  As a result, enzymes have a large molecular weight.  The requirement for multiple weak interactions to drive catalysis is one reason why enzymes (and some coenzymes) are so large. Cont’d  The biocatalyst property of enzymes:  they speed up a reaction without being used up, so they can be used over and over again.  They are effective in small amount. A small amount of enzyme can bring about a change in a large amount of its substrate.  Enzyme precipitation :  is the separation of enzymes for analysis using different aqueous or ethanol solvents.  Because enzymes are amphoteric, they can be precipitated by acidic and alkaline solutions.  The presence of ethanol as well as a high concentration of inorganic salts such as ammonium sulphate aid in the precipitation of enzymes.  Enzymatic activity:  is the general catalytic properties of an enzyme.  The mechanism of enzyme action is highly depend on parameters such as :  temperature, pH, and  enzyme concentration and substrate concentration.  Inhibitors  Enzymes show the highest activity at optimum temperature and pH.  The enzymatic reaction will be slowed down if the enzyme and substrate concentrations are too low. B. Chemical properties of enzymes Cont’d  Heat and PH Sensitivity  Enzymes are affected by pH and temperature.  Work at optimum conditions.  Regulation :  is the process of controlling the activity of enzymes by activator and inhibitor molecules.  Catalysis :  the process of the acceleration of a chemical reaction.  They can transform about 100-10,000 substrates per second.  the reactions catalyzed by the enzymes show a 103-108 times faster reaction rate in comparison to the non-catalyzed reactions.  Reversibility:  the ability of enzymes to catalyze various metabolic (anabolic and catabolic) reactions.  Enzymes catalyze biochemical reactions in both forward and reverse directions.  Enzyme specificity :  Describes how restrictive the enzyme is in its choice of substrate.  A completely specific enzyme would have only one substrate.  catalyzing only one type of chemical reaction.  They are very specific for both the type of reaction they catalyze, and substrate.  Why are enzymes specific? Because of the conformation of the active site.  The specificity is détermine by the active site.  Only substrates with the correct shapes can be bound by the enzyme, minimizing the frequency of side reactions.  Most enzymes are stereospecific and act only on one stereoisomeric form of the Cont…  There are four distinct types of specificity:  1. Substrate specificity:  enzyme acts only on a particular substrate.  2. Group specificity:  a structural specificity of enzymes,  the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate and methyl groups.  3. Bond/Linkage/ specificity:  the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure.  4. Optical /Stereochemical/specificity:  the enzyme will act on a particular steric or optical isomer  enzymes act on the substrate optical configuration.  5. Co-factor specificity  specificity to the substrate and co-factors. Cont…  Enzymes are well suited to their roles in three major ways:  they have enormous catalytic power,  they are highly specific in the reactions they catalyze, and  their activity as catalysts can be regulated. Class work  Why are several enzymes needed in a typical metabolic pathway?  Describe the function of active site of an enzyme?  The Function Of Enzymes  Enzymes are essential for respiration, digesting food, the liver, muscle, and nerve function.  Enzymes are used as markers of disease by measuring their activity in the body, which can indicate tissue damage or disease.  Enzymes are markers of the states of various diseases like :  myocardial infraction:  commonly known as a heart attack, occurs when blood flow decreases or stops in one of the coronary arteries of the heart.  Jaundice:  is a yellow color of the skin, mucus membranes, or eyes.  The yellow coloring comes from bilirubin, a byproduct of old red blood cells.  Jaundice can occur if:  Too many red blood cells are dying or breaking down and going to the liver.  The liver is overloaded or damaged.  The bilirubin from the liver is unable to move normally into the digestive tract.  Jaundice is often a sign of a problem with the liver, blockage gallbladder, or pancreas.  Pancreatitis:  a condition that causes inflammation of the pancreas  cancer and  neurodegenerative disorders etc. Cont’d  Examples of enzymes:  Sucrase - breaks down a sugar called sucrose.  Lactase - breaks down lactose, a kind of sugar found in milk products.  Carbohydrase- breaks down carbohydrates into sugars.  Lipase - breaks down fats into fatty acids.  Protease - breaks down protein into amino acids Cont’d Table 3.1 Some of enzymes in the body and their functions Enzyme Function Lipases Split fats found in the blood, gastric juices, pancreatic secretions, intestinal juices, adipose (fatty) tissues and participate in digestions. Amylase Amylase exists in saliva and helps in changing starches into sugars. Maltase exists in foods such as potatoes, pasta and beer and saliva Maltase breaks the sugar maltose into glucose. Trypsin Found in the small intestine, breaks down of protein into peptide. Found in the small intestine, breaks lactose, the sugar in milk, into Lactase glucose and galactose. Helicase Unwinds DNA An enzyme responsible for forming new copies of DNA by joining DNA Polymerase nucleotides. Breaks down the neurotransmitter acetylcholine in nerves and Acetyl cholinesterase muscles  cont’d  How enzymes work? Mechanisms of Enzyme Catalysis.  The activation energy (or Ea):  the energy required to start off a chemical reaction.  a higher activation energy corresponds to a slower reaction.  the lower activation energy, the more molecules have sufficient energy to pass through the transition state, and, thus, the faster the rate of the reaction.  Catalysts enhance reaction rates by lowering activation energies.  The role of enzymes is to accelerate the inter- conversion of S and P by lowering activation energy. Cont….  For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state. Fig. 3.5 Activation energy for unanalyzed reaction and the same reaction with a catalyst Cont’d…  Mechanisms of Enzyme Catalysis:  Three mechanisms by which enzymes accelerate reactions: ◦ 1. maintaining precise substrate orientation ◦ 2. changing substrate reactivity by altering its electrostatic structure.  enzymes cannot change the pH of their medium, they do contain numerous amino acids with acidic or basic side chains. These groups are capable of donating or accepting protons to and from the substrate, thereby altering the electrostatic character of the substrate, making it more reactive. ◦ 3. exerting physical stress on bonds in the substrate to be broken- Inducing Strain in the Substrate.  Enzyme-substrate binding models  There are two different of enzyme-substrate binding models or models of enzyme action. These are:  Lock-and-key model, first proposed in 1894 by a German biochemist named Fischer.  Induced-fit model, proposed in 1958 by Koshland.  Both models suggest that the enzyme catalyzes the reaction by lowering the activation energy.  However, they differ in the way:  that they explain how this happens.  explaining how the substrate binds to the active site of the enzyme.  Lock and key model  It proposes that the shapes of the substrate molecules are complementary to that of the active site.  The useful way of thinking the model is to think of an egg sitting in an egg cup because the shapes are complementary.  The model explains on how the enzymes must bind to substrates before they catalyze a chemical reaction.  A key is complementary to that of the lock it fits.  It explains the high specificity of enzyme activity.  The complementary substrate molecule binds with the active site of the enzyme to form the enzyme–substrate complex.  The complex causes the reactants to enter a transition state in which the activation energy of the reaction is lowered. The reaction takes place and the products formed are released.  This lock-and key–model of enzyme action suggests that the enzyme lowers the activation energy by providing the alternative pathways for the reaction. Cont…  For example:  Non-catalyzed pathway: ◦ Reactant A + Reactant B ➞ Product AB  Enzyme-catalyzed pathway: ◦ Reactant A + Reactant B + Enzyme ➞ Intermediate ➞ Product AB + Enzyme  This model sees the enzyme–substrate complex as the intermediate, which is part of a pathway that requires less energy than the normal pathway.  However, a weakness of this model is that it does not explain how the intermediate reduces activation energy.  Fig. 3.9 - The Lock-and-Key Model  Induced fit model  This model suggests that the active site and the substrate aren’t naturally complementary in shape, but the binding of substrate molecules produces a conformational change in the enzyme active site, which either enhances or suppresses the activity of the enzyme.  Enzymes have flexible conformations. This allows the substrate and active site to bind fully.  The conformational change also puts the substrate molecules under tension, so they enter ‘a transition state’  So, bonds in the reactants are put under strain and break more easily and rejoin with other bonds to form the products. This is because of the lowered activation energy.   Enzymatic transition state  It describes how the chemical reactions are taking place qualitatively in the activated enzyme- substrate complex of absolute reaction rates.  The reactive state of substrate binding catalysis is corresponding to the maximum reaction activated and its state of transition.  Binding equilibrium does not exist at the transition state because the transition state is too short-lived.  The transition state complex exists only for the duration of bond breaking or forming.  The transition state has the highest free energy, making it a rare and unstable intermediate.  An enzymes helps catalyze a reaction by lowering the free energy of the transition state and stabilizing its structure.  Enzymes don’t affect the free energy of the reactant or products.  Enzymes bind to the substrate to form E-S complex, which lowers the activation energy required for the reaction.  Binding energy is a major source of free energy used by enzymes to lower the activation energies of reactions.  Substrate is converted into product when the substrate has enough energy to overcome the Figure 3.12 Enzymatic activation energy.  transitional states Transition state is central to understanding enzymatic catalysis because enzymes lower the activation energy by binding tightly to the transition state structure.  Turnover and Efficiency of enzymes  The rate of a chemical reaction is the rate at which reactants are converted into products.  In the case of enzyme- controlled reaction, this is determined by how many molecules of substrate bind with enzyme molecules to form enzyme-substrate complexes.  Turnover number(TON):  a measure of enzyme efficiency.  Is a central parameter for quantitative studies of enzymatic activities.  The turnover number is defined as:  is the number of substrates molecules converted by one enzyme molecule per second at saturated (fully occupied) active sites.  is the number of the molecules produced per catalytic site per unit time before deactivation under a given reaction conditions.  Catalase has the highest turnover numbers of all enzymes.  One molecule of catalase can convert over 2.8 million molecules of hydrogen peroxide to water and oxygen per second.  In a distant second place, carbonic anhydrase catalyzes CO2 to form bicarbonate at the rate of 600,000 molecules per second.  Most other enzymes have a turnover numbers in the range of 1-10,000.  Without enzymes as catalysts, many biochemical reactions would take years rather than seconds.  Enzyme regulation/regulatory enzyme/  What are the enzyme regulations?  Enzyme regulation is a control system for enzymatic activities in which enzymes are turned “on” or “off” depending on the organisms need.  Enzyme regulation occurs by the addition or elimination of some molecules attaching to the enzyme.  Regulatory enzymes exhibit increased or decreased catalytic activity in response to certain signals. It requires an extra activation process to pass through some modifications and function. In most multienzyme systems, the first enzyme of the sequence is a regulatory enzyme.  The activities of regulatory enzymes are modulated by: ◦ 1. Allosteric enzymes :  are enzymes that have additional binding sites for effector molecules other than the active site that cause conformational changes, leading to changes of catalytic properties.  contain two binding sites called active site/catalytic site and allosteric site/regulatory site for binding substrates and Figure 3.13 Allosteric enzyme effectors respectively. Cont’d  Effectors or allosteric modulators are small metabolite molecules or cofactors(inhibitor or activator) modulating the enzyme activity.  Effectors or allosteric modulators function through reversible non-covalent binding of a regulatory metabolite in the allosteric site or non-active site.  Effectors lead to conformational changes in a concrete part of the enzyme that affect the overall conformation of the active site, causing modifications in the activity of the reaction  2. Genetic and covalently modulated enzymes: ◦ The genetic and covalent modification modifies the protein surface and facilitates intracellular delivery. ◦ Genetic modification of enzymes is to improve the properties of enzymes and gain active and inactive forms. ◦ Covalent modulated enzymes are active and inactive forms of the enzymes altered due to covalent modification of structures catalyzed by other enzymes. ◦ Covalently modulated enzymes are regulated by reversible covalent modification of a specific functional group necessary for activity. ◦ The phosphorylation of specific amino acid residues is a particularly common way to regulate enzyme activity. ◦ Modifications can target a single type of amino acid or multiple amino acids and will change the chemical properties of the site. Cont’d  Phosphorylation is the addition of phosphate groups to proteins.  It is the most frequent regulatory modification mechanism in our cells. Cont’d  The properties of allosteric enzymes are significantly different from those of simple non- regulatory enzymes.  Some of the differences are structural.  Allosteric enzymes are generally larger and more complex than non-allosteric enzymes.  in addition to active sites, allosteric enzymes generally have one or more regulatory, or allosteric, sites for binding the modulator.  Just as an enzyme’s active site is specific for its substrate, each regulatory site is specific for its modulator.  In homo-tropic enzymes, the active site and regulatory site are the same.  Often the modulator is the substrate itself; regulatory enzymes for which substrate and modulator are identical are called homo-tropic  Types of enzymes  Enzyme structural classification:  an enzyme classified into simple proteins (active) and conjugated proteins (holoenzymes).  Cont…  Apoenzyme(inactive protein)-a protein portion that combines with a cofactor, to form an active enzyme.  Cofactor- a small, non-protein particle essential for the activity of some enzymes.  The cofactor combines with the apoenzyme to produce an active enzyme, i.e. holoenzyme.  Cont’d..  Cofactors :  Small, non protein molecule that essential for enzymatic catalysis.  They bind directly to the enzyme surface forming an active site for the substrate to bind or interact.  They serves as transient active site.  Cofactors, unlike enzymes, are able at relatively high temperature/thermostable/.  Co-factors either remain unchanged at the end of reaction.  They can regenerated on each turnover of substrates.  They can be reversibly dissociated from the enzyme.  Neither apoenzyme nor the cofactors alone have catalytic activity.  cont’d  The role of cofactor are both structural and functional.  They promote catalytic activity or stability of an enzyme.  They providing electrophilic site to interact with substrate.  They provide the enzyme with the chemical or photochemical capabilities lacking in the normal amino acid side chain.  They aid in substrate binding, catalysis, stabilizing the transition state or contributing to the overall stability of the enzyme structure  cofactors include:  Coenzymes  Mineral ions  Prosthetic groups  Coenzymes-are non-protein organic molecules.  Most are derived from water soluble B-vitamins and give catalytic activity.  loosely bound to the enzyme.  Are thermostable  Some coenzymes serve as transient carriers of specific atoms or functional groups Common coenzymes and their functions Coenzyme Vitamin Enzyme Function Nicotinamide Niacin(Vitamin Oxidoreductase Oxidation or adenine B3) in respiration hydrogen transfer dinucleotide in respiration (NAD) Flavin adenine Riboflavin Oxidoreductase Oxidation or dinucleotide (vitamin B2) in respiration hydrogen transfer (FAD) in respiration Cont’d  Mineral ions- bind loosely with the enzyme to give it its catalytic activity.  Includes K+, Fe++, Fe+++, Cu++, Co++, Zn++, Mn++, Mg++, Ca++, and Mo+++. Table 3.6 Enzymes that require mineral ions as cofactors Cont…  Prosthetic group: thermostable  A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme protein is called a prosthetic group.  Examples include, flavin mononucleotide (FMN), heme and biotin.  Some enzymes require both a coenzyme and one or more metal ions for activity.  Basic classification of Enzymes  It is introduced by the International Union of Biochemistry (I.U.B.) in 1961.  Enzymes has systematic name that specifies :  the substrate of the enzyme (the substance acted on),  the functional group acted on, and the type of reaction catalyzed.  In the systematic naming system, enzymes are divided into six major classes on the basis of:  the reaction which they catalyze and the end suffix “-ase”  on what and how they react  The systematic names are unambiguous and informative. Table 3.2- Classification of enzymes Cont…  Each class of enzymes contains several different, but related, subclasses.  Each subclass is further divided into sub-subclasses.  Within the sub-subclasses, each enzyme has a number.  In the systematic naming of enzymes, an enzyme will have a ‘name’ such as EC 3.4.11.1  EC stands for Enzyme Commission  3- stands for main class the enzyme belongs(Hydrolases)  4- indicates a subclass(tells enzyme action & clue about substrate)  11- indicates a sub-subclass(tell us nature of bond)  1- is the serial number of the type enzyme in its sub subclass. Cont…  Enzyme EC 3.4.11.1 is:  A hydrolase – all the enzymes in class three hydrolyze some kind of bond.  A peptidase – all the enzymes in subclass 4 of class 3 are peptidase and hydrolyze peptide bonds  An amino-peptidase – all the enzymes in sub–subclass 11 of sub-class 4 are amino- peptidases; they hydrolyze peptide bonds at the amino end of a polypeptide chain  Leucyl-amino-peptidase – this particular amino-peptidase is number 1 of this sub- subclass  Home work  visit site: ◦www.chem.qmul.ac.uk/iubmb/ enzyme/ and find out more about the naming of enzymes. What is enzyme 1.1.1.1? Factors affecting the enzymes action  Temperature  Optimum Temperature : ◦ all enzymes work best within the specific ranges of optimum temperatures. ◦ Temperature at which an enzyme works most efficiently.  Greatest number of molecular collisions.  ‘Free’ particles move around more quickly.  ↑temperature, ↑kinetic energy leads more molecular collision between enzyme and substrate.  Human enzymes = 35°- 40°C  Enzymes do not all have the same optimum temperature.  They are adapted to work most efficiently within the organism in which they are found.  For example, the optimum temperature for enzymes:  Organism optimum temperature ◦ In human beings - 37 °C ◦ in plants growing in the Arctic - < 5 °C ◦ thermophilic bacteria - > 90 °C. Cont’d Different enzymes function in different organisms in different environments.  Cont’d  low or high temperature causes an enzyme to lose its activity and ability to bind a substrate and denatured. Once enzymes denatured, they cannot be renatured.  Increase T° beyond optimum T°: ◦ Particles within a molecule vibrate more energetically. ◦ Increased energy level of molecules disrupts or strains on bonds in enzyme & between enzyme & substrate. ◦ Bonds begin to break. ◦ The enzyme begin to lose its tertiary structure and denature mainly by breaking H-bonds and causes it to lose its normal biological activity. ◦ Heat-induced denaturation is not usually reversible.  Cont’d cold: decrease T° below optimum:  Molecules move slower  decrease collisions between enzyme & substrate  Enzymes become inactive  The PH scale  PH is a measure of the acidity /alkality or hydrogen ion concentration of a solution.  It is measured on a scale of 0-14 with pH values.  below 7 being acidic,  values above 7 being alkaline and  a value of 7 being neutral.  The highest H+ concentration and is the most acid.  The lowest H+ concentration and is the most alkaline  As the pH changes away from the optimum toward the acidic range of the pH scale, an enzyme tends to gain hydrogen ions from the solution.  As the pH changes away from the optimum toward the alkaline range of the pH scale, the enzyme tends to lose hydrogen ions to the solution. Cont’d  In both cases, changes are produced in the weak interactions that maintain the shape of the enzyme molecule and the charge of amino acids that form the active site.  Significant changes in PH can affect an enzyme molecule by:  Breaking ionic bond that hold the tertiary structure in place and denature enzymes.  PH-induced denaturation is usually reversible.  Altering the charge of amino acids that form the active site.  Therefore substrate can no longer bind to the active site and so enzyme action decreases. Cont’d  Optimum PH:  Enzyme action is greatest within a narrow range of pH, because all enzymes are active.  Each enzyme has an optimum pH that works more efficiently.  Above or below an enzyme’s optimum pH, its activity is lower.  The optimum pH of a particular enzyme corresponds to the pH of its natural environment or depends on localized conditions.  For many enzymes, this corresponds to pH values of around 7.  Majority of enzymes in most mammals function most efficiently within the PH range of 6-8. Cont’d  The PH of human gut enzymes :  Human gut region Enzymes optimum PH  Mouth Salivary amylase b/n 5 & 7.5  Stomach Pepsin PH 1.5 and 3.  Small intestine Trypsin PH 8  PH in the mouth varies from being slightly alkaline(PH 7.5) to acidic(PH5)  Depends on whether or not we have eaten and also what we have eaten.  The PH in the stomach can be as low as PH 1.5.  The PH of small intestine is slightly alkaline at PH 7.5  Cont’d Figure 3.16 The optimum pHs of some human digestive enzymes Human intracellular enzymes work best at 37°C and pH 7.  Substrate concentration  At low substrate concentration the reaction proceeds slowly. This is because:  there are no enough substrate molecules to occupy all of the active sites on the enzyme.  As substrate concentration increases, the reaction rate increase and more enzyme substrate complexes formed.  If the concentration of enzymes remains constant, increasing the substrate concentration increases the rate of reaction until all the active sit are occupied/saturated.  As ↑ substrate = ↑ reaction rate  more substrate = more frequently collide Cont’d…  Each enzyme molecule could be working at maximum turnover, so the activity of enzyme is likely to remain constant.  Reaction rate levels off:  When all of enzymes active site are engaged/occupied or enzyme is saturated by substrate.  maximum rate of reaction  Enzymes concentration  At low enzyme concentrations there are more substrate molecules than there are available active sites.  Increasing the number of active sites by increasing the concentration of the enzyme, therefore, effectively increases the rate of the reaction.  At constant large supply of substrate molecules, each enzyme molecule will work at maximum turnover. Therefore, the reaction rate will increase. Because the reaction rate will be directly proportional to the concentration of the enzyme.  as ↑ enzyme = ↑ reaction rate  more enzymes = more frequently collide with substrate  However, increasing the concentration of the enzyme will not increase the activity of the enzyme.  Each enzyme molecule will be working at maximum turnover, so the activity of the enzyme is likely to remain constant. Cont’d  If the substrate concentration is high and constant, increasing the enzyme concentration increases the reaction rate.  Reaction rate levels off:  When substrate molecule becomes limiting factor therefore, increasing concentration of the enzyme will not increase the activity of the enzyme or rate of reaction. Cont’d  Radiation :  damages enzyme activities by reducing in enzymatic efficiency and creating disorders in the macromolecules.  Water:  It affects enzyme action in several ways:  Enzyme stability: water helps enzymes maintain their natural conformation and stability.  Enzyme solubility: water helps enzymes dissolve and move freely to reach substrates.  Enzyme activity: too much or too little water can negatively affect the performance of enzymes’ activity.  Enzyme inhibition  Enzyme Inhibitors are substances that bind to enzymes and prevent enzymes from forming enzyme–substrate complexes and, as a result, stop, or slow down, the reaction.  They are usually specific and they work at low concentrations  They block the enzyme but they do not usually destroy it  There are two main types enzyme inhibitors. These are :  reversible inhibitors  irreversible inhibitors Cont’d  Irreversible inhibitors  Bind tightly and permanently to enzymes, usually by a covalent bond.  is a substance that permanently blocks the action of an enzyme.  They work at very low concentration of inhibitors.  Examples:  1. the painkiller aspirin binds with the enzyme cyclooxidase-2, which is an important enzyme in producing prostaglandins which give the sensation of pain.  Prostaglandin is an unsaturated fatty acid found in all mammals that performs a similar function to that of hormones in controlling smooth muscle contraction, blood pressure, inflammation, and body temperature. Cont’d  2. Very small concentration of heavy metal ions such as:  Mercury(Hg2+)  Silver(Ag+)  Arsenic (As+) bind with (-SH) groups in the active site.  Lead(Pb2+)  3. The nerve gas DFP(Diisopropylfluoro phosphate)-  Used in warfare  It combines with serine amino acid at the active site of enzyme acetyl cholinesterase.  This enzyme deactivates the acetylcholine(neurotransmitter). Cont’d  If acetylcholinesterase is inhibited, acetylcholine accumulates and nerve impulses can not be stopped, causing prolonged muscle contraction.  Paralysis occurs and death may results since the respiratory muscles are among those affected.  4. a number of insecticides and drugs  Enzymes themselves can act as poisons if they get into the wrong compartment of the body.  Reversible inhibitors  Bind only weakly to enzymes and the bond that holds them breaks easily releasing the inhibitor.  Inactivates an enzyme through non-covalent easily reversed interactions.  Temporarily alter the structure of enzyme.  When inhibitor is removed, the enzyme activity is restored.  There are three main kinds of reversible inhibitors:  Competitive inhibitors  Non-competitive inhibitors  Uncompetitive inhibitors  Competitive reversible inhibition  is a molecule that blocks the binding of the substrate to the active site.  Competes with the substrate for the active site of an enzyme.  Their molecules have shapes which are complementary to all, or part, of the active site of an enzyme.  They are often similar in shape to the substrate molecules.  They can bind with the active site and prevent substrate molecules from binding.  The binding is only temporary and does not permanently damage the enzymes. Cont’d  The overall effect on the rate of reaction depends on the relative concentrations of substrate and inhibitor molecules.  Examples: If there were 99 substrate molecules for every inhibitor molecule then:  99% of the collisions would be between E and S.  The reaction would proceed at 99% of the maximum rate.  If the ratio were 90 substrate molecules to ten inhibitor molecules, there would be:  10% inhibition  The reaction rate would fall to 90% of maximum. Cont’d  Increasing substrate concentration effectively ‘dilutes’ the effect of the inhibitor.  If enough substrate is added, the inhibitor is unlikely to collide with the enzyme. Fig. 3.18 Effect of substrate concentration on inhibition by a competitive inhibitor Cont’d  Examples:  *The painkiller ibuprofen act on the enzyme cyclo-oxidase-2, competing with the precursors of prostaglandins.  *Cyanide (metabolic poison) acts as a competitive inhibitor of the enzyme cytochrome oxidase, an important enzyme in the release of energy in respiration.  The action of sulphonamides(the first antibiotics).  Inhibiting an enzyme needed for the synthesis of folic acid.  Non-competitive inhibitors  Do not compete with the substrate for the active site. Instead, they bind to the allosteric site.  Have no structural similarity to substrate.  Produces a conformational change in the enzyme & its active site.  The active site can no longer bind with the substrate to catalyze the reaction.  The effectiveness of non-competitive inhibitors is not affected by the concentration of the substrate.  Suppose there are enough inhibitor molecules to bind with the allosteric sites of 80% of the enzyme molecules, the non- competitive inhibition bind with their allosteric sites.  80% of the enzyme will be inhibited irrespective of the number of substrate molecules and  The reaction rate will drop to 20% of maximum.  Non-competitive inhibitors- are particularly important in regulating metabolic pathways in cells. Fig. 3.20 The effect of substrate concentration on non-competitive inhibitor  Uncompetitive inhibitor  unlike a competitive inhibitor, binds only to the enzyme substrate complex, but not to the free enzyme.  It occurs in reactions with two or more substrates or products and slows enzyme reactions by binding the substrate to each other.  Can not be overcome by increasing substrate concentration. Cont’d Table 3.7 Enzyme inhibition Substrate Inhibitors Enzymes Inhibition Products Precursor of Aspirin Cyclo-oxidase- Irreversible inhibition Prostaglandins prostaglandins 2 Precursor of Ibuprofen Cyclo-oxidase- Competitive inhibition Prostaglandins prostaglandins 2 Intermediate-A Cyanide Cytochrome Competitive inhibition Intermediate-A oxidase (ATP is released) Threonine Isoleucine Threonine Non-competitive Isoleucine deaminase inhibition(allosteric inhibition) Class work  Which of the following represents the correct interpretation of the graph:  A. X is with the competitive inhibitor, Y with the noncompetitive inhibitor and Z with no inhibitor  B. X is with the non-competitive inhibitor, Y with the  competitive inhibitor and Z with no inhibitor  C. X is with no inhibitor, Y with the non-competitive inhibitor  and Z with the competitive inhibitor  D. X is with no inhibitor, Y with the competitive inhibitor and Z with the non-competitive inhibitor  CLASS WORK  2. Suppose 25% of the molecules of an enzyme are inhibited by a non-competitive inhibitor, which one of the following would happen if the amount of the substrate is increased by 50%? A. The reaction rate would be double B. More enzyme molecules would get inhibited C. The rate of the reaction would decrease by 50% D. The rate of the reaction would remain unchanged  END PRODUCT INHIBITION(FEEDBACK INHIBITION)  is a cellular control mechanism in that the end product inhibit enzyme's activity.  In feedback inhibition, the end product binds to the allosteric site of the enzyme and change the structure of the active site. This prevents the enzyme to perform its activity.  Due to feedback inhibition, a cell is able to know whether the amount of a product is enough for its subsistence or not.  The first step (controlled by E1 is often controlled by the end product (E.  Therefore negative feedback is possible.  A→ B→ C → D→ E  The end products are controlling their own rate of production.  Final product inhibits the enzyme controlling the first stage of a reaction sequence.  If the requirement for substance E in the cell decreases, then the concentration of E will increase. These may be potentially high concentration could be toxic.  Substance E acts as non-competitive inhibitor, which prevents enzymes catalysing the reaction that converts A to B. As result, the entire reaction sequence is halted/stop.  There is no build-up of intermediates (B, C, D)  no unnecessary accumulation of product . Example: The drug Tipranivir used to treat HIV blocks the activity of a viral genome enzyme to make more copies as a reversible inhibitor. Class work  1. Which of the following types of enzyme inhibitions can be removed when the end product of the metabolic pathway is depleted?  A. Allosteric inhibition C. Competitive inhibition  B. Non-reversible inhibition D. Reversible competitive inhibition  Experiment on the functions of enzymes  The enzyme catalase is commonly used in these sorts of investigations. This is because  it is found in almost all cells and there are many readily available sources that contain significant amounts of catalase.  These include: bacteria, yeast, liver(animals) and potato(plants).  It is an important enzyme that protects cells from oxidative damage by reactive oxygen.  Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen. The equation for the reaction is:  Because oxygen is a gas, the volume of oxygen collected in a certain time is a measure of how fast the reaction is proceeding. Cont’d Table 3.5 Improving the investigation Factor How controlled Note controlled Use of water bath Stand the potato pieces in the conical flask and Temperature at the required hydrogen peroxide in the water bath separately temperature for 10 minutes. This is called equilibration. Use of buffer Buffer solutions resist changes in pH and pH solutions at maintain a more or less constant pH. Add the required pH buffer solution to the potato pieces at the start. Carrying out the experiment more than once Carry out the allows us to spot anomalous results and eliminate Repeats experiment three them. This is easier if you have an odd number of or five times results.  Enzyme kinetics  How do you measure the enzymatic rates of reactions?  Enzyme kinetics describes the rates of chemical reactions that are catalyzed by enzymes and the binding affinities of substrates, inhibitors and the maximal catalytic rates achieved.  Enzyme kinetics explains that enzymes speed up reactions by lowering the activation energy of the reactants and turning them into products.  The concentration of enzyme and substrates determines the rate of the reactions or production volumes per unit time.  The relationship between the rate of reaction and concentration of substrate depends on the affinity of the enzyme for its substrate. This is usually expressed as the Km(Michaelis constant) of the enzyme , an inverse measure of affinity. The Michaelis-Menten Model on Enzyme Kinetics  It is one of the most known models of enzyme kinetics.  The Michaelis-Menten formula describing the rate of enzymatic reaction by relating the reaction rate, rate of formation of product to the concentration of substrate.  The model explains the relationship between the rate of an enzyme-catalyzed reaction [V1], the concentration of substrate [S] and two constants, Vmax and Km with the following equation.  Where,  V1 = the initial velocity/rate reaction  Vmax = the maximal velocity/maximum rate of reaction  [S] = the substrate concentration  KM = substrate concentration at half-maximal velocity (Michaelis constant) Cont’d  The rate of reaction when the enzyme is saturated with substrate is the maximum rate of reaction, Vmax.  Km is the concentration of substrate which permits the enzyme to achieve ½ Vmax.  An enzyme with a high Km has a low affinity for its substrate, and requires a greater Figure3.22 Enzyme kinetics as a concentration of function of concentration substrates to achieve Vmax. The effects of inhibitors on enzyme kinetics.  Enzymes vary greatly in their ability to catalyze reactions.  The catalytic activity of an enzyme is revealed by studying its kinetics, that is, the rate at which it catalyzes a reaction under various experimental conditions.  In 1913, Leonor Michaelis and Maud Menten reported on the mathematical relationship between substrate concentration and the velocity of enzyme reactions as measured by the amount of product formed (or substrate consumed) in a given amount of time. Cont’d  According to the equation, when the substrate concentration [S] is set at a value equivalent to KM, then the velocity of the reaction (V) becomes equal to Vmax/2, or one-half the maximal velocity. Thus, KM = [S], when V = Vmax/2. Cont’d  Competitive inhibitor  The affinity of the substrate appears to be decreased when inhibitor is present  (K m,app >Km)  Competitive inhibitors alter the apparent Km, not the Vmax  prevents substrate from binding, so increases Km and no effect on Vmax  Vmax,app = Vmax Km,app > Km  Competitive inhibitor: Increases km, but has no effect on vmax. Cont’d Reaction Rate - Inhibitor Vmax + Inhibitor Vmax 2 Km Km,app [Substrate] Cont’d  Non-competitive inhibitors :  The maximum velocity appears to be decreased in the presence of the inhibitor (Vmax,app