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Enzyme And Metabolism


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Enzyme And Metabolism

  2. 2. OBJECTIVES: <ul><li>By the end of the lesson, student should be able to : </li></ul><ul><li>Define terms enzyme. </li></ul><ul><li>Understand the Lock & Key model and Induced-fit model. </li></ul><ul><li>Identify the groups of enzymes. </li></ul><ul><li>Understand some factors affecting the enzyme activities. </li></ul>
  3. 3. <ul><li>Enzymes are proteins that catalyze ( i.e. accelerate) and control the rates of chemical reactions. </li></ul><ul><li>In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products. </li></ul><ul><li>Almost all processes in a biological cell need enzymes in order to occur at significant rates. </li></ul>ENZYME??
  4. 4. <ul><li>Since enzymes are extremely selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell. </li></ul>
  5. 5. ENZYME AS BIOLOGICAL CATALYSTS: <ul><li>Enzymes are biological catalysts produced by living cells. </li></ul><ul><li>Enzymes lower the amount of activation energy needed. </li></ul><ul><li>They speed up the rate of biochemical reactions in the cell but remain unchanged at the end of the reactions. </li></ul><ul><li>Most enzymes are globular protein molecules. </li></ul>
  6. 6. <ul><li>The chemicals which an enzyme acts on is called its substrate. </li></ul><ul><li>The enzyme combines with its substrate to form an enzyme-substrate complex. </li></ul><ul><li>The complex than breaks up into product and enzyme. </li></ul><ul><li>A metabolic pathway is a number of reactions catalysed by sequence of enzymes. </li></ul>
  7. 8. MECHANISM ACTION: There are 2 main hypotheses explaining of enzyme action. Lock & Key model Induced - fit model
  8. 9. <ul><li>Each enzyme is specific for one and ONLY one substrate (one lock - one key) </li></ul><ul><li>active site : part of the enzyme that fits with the substrate </li></ul><ul><li>Note that the active site has a specific fit for this particular substrate and no other. </li></ul><ul><li>This theory has some weaknesses, but it explains many basic things about enzyme function.  </li></ul>LOCK & KEY MODEL
  9. 10. <ul><li>Substrate: The starting molecules for a chemical reaction are called the substrates. </li></ul><ul><li>Enzyme substrate complex: The enzyme substrate complex is transitional step when the substrates of a chemical reaction are bound to the enzyme. </li></ul><ul><li>Active site: The area on the enzyme where the substrate or substrates attach to is called the active site. </li></ul><ul><li>Enzymes are usually very large proteins and the active site is just a small region of the enzyme molecule. </li></ul>
  10. 14. <ul><li>The induced-fit theory assumes that the substrate plays a role in determining the final shape of the enzyme and that the enzyme is partially flexible. </li></ul><ul><li>This explains why certain compounds can bind to the enzyme but do not react because the enzyme has been distorted too much. </li></ul><ul><li>Other molecules may be too small to induce the proper alignment and therefore cannot react. </li></ul><ul><li>Only the proper substrate is capable of inducing the proper alignment of the active site. </li></ul>INDUCED-FIT HYPOTHESIS
  12. 16. <ul><li>In the graphic, the substrate is represented by the magenta molecule, the enzyme protein is represented by the green and cyan colors. </li></ul><ul><li>The cyan colored protein is used to more sharply define the active site. </li></ul><ul><li>The protein chains are flexible and fit around the substrate. </li></ul>INDUCED-FIT HYPOTHESIS
  13. 18. <ul><li>The advantages of the induced fit mechanism arise due to the stabilizing effect of strong enzyme binding. </li></ul><ul><li>There are two different mechanisms of substrate binding; uniform binding which has strong substrate binding, and differential binding which has strong transition state binding. </li></ul><ul><li>The stabilizing effect of uniform binding increases both substrate and transition state binding affinity and differential binding increases only transition state binding affinity. </li></ul>
  14. 19. <ul><li>Both are used by enzymes and have been evolutionarily chosen to minimize the ΔG of the reaction. </li></ul><ul><li>Enzymes which are saturated, ie. have a high affinity substrate binding, require differential binding to reduce the ΔG, whereas largely substrate unbound enzymes may use either differential or uniform binding. </li></ul>
  15. 21. How do enzymes work? <ul><li>substrate : molecules upon which an enzyme acts. The enzyme is shaped so that it can only lock up with a specific substrate molecule. </li></ul><ul><li>enzyme </li></ul><ul><li>substrate -------------> product </li></ul> 
  16. 23. <ul><li>The diagram shows time on the horizontal axis and the amount of energy in the chemicals involved in a chemical reaction on the vertical axis. </li></ul><ul><li>The point if this diagram again is that without the enzyme, much more activation energy is required to get a chemical reaction to take place. </li></ul>
  17. 26. Factors Influencing Enzyme Activity <ul><li>pH : the optimum (best) in most living things is close to 7 (neutral). </li></ul><ul><li>High or low pH levels usually slow enzyme activity </li></ul>
  18. 27. <ul><li>Temperature : strongly influences enzyme activity optimum (best) temperature for maximum enzyme function is usually about 35-40 C. </li></ul><ul><li>Reactions proceed slowly below optimal temperatures. </li></ul><ul><li>Above 45 C. most enzymes are denatured (change in their shape so the enzyme active site no longer fits with the substrate and the enzyme can't function) </li></ul>
  19. 30. METABOLISM <ul><li>Metabolism is the sum of all biochemical reactions occurring in living cells. </li></ul><ul><li>These reactions can be divided into two main groups: </li></ul><ul><ul><li>1) ANABOLISM </li></ul></ul><ul><ul><li>2) CATABOLISM </li></ul></ul>
  20. 31. <ul><li>Involves the synthesis of complex molecules from simpler molecules which requires energy input. </li></ul><ul><li>Involves the breakdown of complex molecules into simpler molecules involving hydrolysis or oxidation and the release of energy. </li></ul>ANABOLISM CATABOLISM
  21. 32. <ul><li>Energy releasing processes, ones that &quot;generate&quot; energy, are termed exergonic reactions. </li></ul><ul><li>Reactions that require energy to initiate the reaction are known as endergonic reactions. </li></ul><ul><li>All natural processes tend to proceed in such a direction that the disorder or randomness of the universe increases </li></ul>EXERGONIC REACTIONS
  22. 33. <ul><li>In an exergonic reaction the change is free energy is represented by a negative number (-G), indicating free energy is released during the reaction. </li></ul>
  23. 35. <ul><li>This kind of reaction is not termed a spontaneous reaction. In order to go from the initial state to the final state a considerable amount of energy must be imparted to the system. </li></ul><ul><li>These kinds of reactions are associated with a positive number (+G). </li></ul>ENDERGONIC REACTIONS
  25. 39. <ul><li>The speed V means the number of reactions per second that are catalyzed by an enzyme. </li></ul><ul><li>With increasing substrate concentration [S], the enzyme is asymptotically approaching its maximum speed V max, but never actually reaching it. </li></ul><ul><li>Because of that, no [S] for V max can be given. </li></ul><ul><li>Instead, the characteristic value for the enzyme is defined by the substrate concentration at its half-maximum speed ( V max /2 ). </li></ul><ul><li>This KM value is also called Michaelis-Menten constant. </li></ul>
  26. 40. <ul><li>Vo = Initial reaction velocity </li></ul><ul><li>V max = Maximum velocity </li></ul><ul><li>K m = Michaelis constant </li></ul><ul><li>[S] = Substrate concentration </li></ul>V o = V max K M
  27. 41. <ul><li>A non protein component of enzymes is called the cofactor. </li></ul><ul><li>If the cofactor is organic, then it is called a coenzyme. </li></ul><ul><li>Coenzymes are relatively small molecules compared to the protein part of the enzyme. </li></ul><ul><li>Many of the coenzymes are derived from vitamins. </li></ul><ul><li>The coenzymes make up a part of the active site, since without the coenzyme, the enzyme will not function. </li></ul>ENZYME COFACTORS
  28. 42. <ul><li>In the graphic on the left is the structure for the coenzyme, NAD+, Nicotinamide Adenine Dinucleotide. </li></ul><ul><li>Nicotinamide is from the niacin vitamin. </li></ul><ul><li>The NAD+ coenzyme is involved with many types of oxidation reactions where alcohols are converted to ketones or aldehydes. </li></ul>ENZYME COFACTORS
  29. 43.   Aldehyde group transfer   thiaminpyrophosphate (TPP)   thiamin (B-1)   Methyl group transfer   coenzyme B-12   vitamin B-12   Acetyl group carrier   coenzyme A (CoA)   pantothenic acid   oxidation or hydrogen transfer   flavin adenine dinucleotide (FAD)   riboflavin   oxidation or hydrogen transfer   nicotinamide adenine dinucleotide (NAD + )   niacin   Function   Coenzyme   Vitamin
  30. 44. <ul><li>Coenzyme Q10 is a fat-soluble nutrient also known as CoQ10, vitamin Q10, ubidecarenone, or ubiquinone. </li></ul><ul><li>It is a natural product of the human body that is primarily found in the mitochondria, which are the cellular organelles that produce energy. </li></ul><ul><li>It occurs in most tissues of the human body; however, the highest concentrations are found in the heart, liver, kidneys, and pancreas. </li></ul><ul><li>Ubiquinone takes its name from a combination of the word ubiquitous, meaning something that is found everywhere, and quinone 10. </li></ul>COENZYMES
  31. 45. <ul><li>Quinones are substances found in all plants and animals. </li></ul><ul><li>The variety found in humans has a 10-unit side chain in its molecular structure. </li></ul><ul><li>Apart from the important process that provides energy, CoQ10 also stabilizes cell membranes and acts as an antioxidant. </li></ul><ul><li>In this capacity, it destroys free radicals, which are unstable molecules that can damage normal cells. </li></ul>
  32. 48. <ul><li>Enzyme inhibitors are molecules that interact in some way with the enzyme to prevent it from working in the normal manner. </li></ul><ul><li>There are a variety of types of inhibitors including: nonspecific, irreversible, reversible - competitive and noncompetitive. </li></ul><ul><li>Poisons and drugs are examples of enzyme inhibitors. </li></ul>ENZYME INHHIBITORS
  33. 50. <ul><li>A nonspecific inhibition effects all enzymes in the same way. </li></ul><ul><li>Non-specific methods of inhibition include any physical or chemical changes which ultimately denatures the protein portion of the enzyme and are therefore irreversible. </li></ul>NON SPECIFIC INHIBITORS
  34. 51. <ul><li>Temperature: Usually, the reaction rate increases with temperature, but with enzyme reactions, a point is reached when the reaction rate decreases with increasing temperature. </li></ul><ul><li>At high temperatures the protein part of the enzyme begins to denature, thus inhibiting the reaction. </li></ul>EXAMPLE:
  35. 52. <ul><li>A competitive inhibitor is any compound which closely resembles the chemical structure and molecular geometry of the substrate. </li></ul><ul><li>The inhibitor competes for the same active site as the substrate molecule. </li></ul><ul><li>The inhibitor may interact with the enzyme at the active site, but no reaction takes place. </li></ul>COMPETITIVE INHIBITORS
  36. 53. <ul><li>The inhibitor is &quot;stuck&quot; on the enzyme and prevents any substrate molecules from reacting with the enzyme. </li></ul><ul><li>However, a competitive inhibition is usually reversible if sufficient substrate molecules are available to ultimately displace the inhibitor. </li></ul><ul><li>Therefore, the amount of enzyme inhibition depends upon the inhibitor concentration, substrate concentration, and the relative affinities of the inhibitor and substrate for the active site. </li></ul>COMPETITIVE INHIBITORS
  37. 56. <ul><li>A noncompetitive inhibitor is a substance that forms strong covalent bonds with an enzyme and consequently may not be displaced by the addition of excess substrate. </li></ul><ul><li>Therefore, noncompetitive inhibition is irreversible. </li></ul><ul><li>A noncompetitive inhibitor may be bonded at, near, or remote from the active site. In any case, the basic structure of the enzyme is modified to the degree that it ceases to work. </li></ul>NON-COMPETITORS INHIBITORS
  39. 58. <ul><li>If the inhibition is at a place remote from the active site, this is called allosteric inhibition. </li></ul><ul><li>Allosteric means &quot;other site&quot; or &quot;other structure&quot;. </li></ul><ul><li>The interaction of an inhibitor at an allosteric site changes the structure of the enzyme so that the active site is also changed. </li></ul>NON-COMPETITORS INHIBITORS
  40. 59. <ul><li>There are approximately 3000 enzymes which have been characterised.  </li></ul><ul><li>These are grouped into six main classes according to the type of reaction catalysed.  </li></ul><ul><li>At present, only a limited number are used in enzyme electrodes or for other analytical purposes. </li></ul>CLASSIFICATION OF ENZYME
  41. 60. 1.Oxidoreductases <ul><li>These enzymes catalyse oxidation and reduction reactions involving the transfer of hydrogen atoms or electrons.  </li></ul><ul><li>The following are of particular importance in the design of enzyme electrodes. </li></ul><ul><li>This group can be further divided into 4 main classes.   </li></ul>
  42. 61. <ul><ul><li>catalyse hydrogen transfer from the substrate to molecular oxygen producing hydrogen peroxide as a by-product.  An example of this is FAD dependent glucose oxidase which catalyses the following reaction: </li></ul></ul><ul><ul><li>  b-D-glucose + O2 = gluconolactone + H2O2 </li></ul></ul>oxidases
  43. 62. dehydrogenases <ul><ul><li>catalyse hydrogen transfer from the substrate to a nicotinamide adenine dinucleotide cofactor (NAD+).  An example of this is lactate dehydrogenase which catalyses the following reaction: </li></ul></ul><ul><ul><li>Lactate + NAD+ = Pyruvate + NADH + H+ </li></ul></ul>
  44. 63. peroxidases <ul><ul><li>catalyse oxidation of a substrate by hydrogen peroxide.  </li></ul></ul><ul><ul><li>An example of this type of enzyme is horseradish peroxidase which catalyses the oxidation of a number of different reducing substances (dyes, amines, hydroquinones etc.) and the concomitant reduction of hydrogen peroxide. </li></ul></ul><ul><ul><li>The reaction below illustrates the oxidation of neutral ferrocene to ferricinium in the presence of hydrogen peroxide: </li></ul></ul><ul><ul><li>2[Fe(Cp)2] + H2O2 + 2H+= 2[Fe(Cp)2]+ + 2 H2O   </li></ul></ul>
  45. 64. <ul><ul><li>catalyse substrate oxidation by molecular oxygen.  </li></ul></ul><ul><ul><li>The reduced product of the reaction in this case is water and not hydrogen peroxide.  </li></ul></ul><ul><ul><li>An example of this is the oxidation of lactate to acetate catalysed by lactate-2-monooxygenase. </li></ul></ul><ul><ul><li>  lactate + O2 = acetate + CO2 + H2O </li></ul></ul>oxygenases
  46. 65. 2.Transferases <ul><li>These enzymes transfer C, N, P or S containing groups (alkyl, acyl, aldehyde, amino, phosphate or glucosyl) from one substrate to another.  </li></ul><ul><li>Transaminases, transketolases, transaldolases and transmethylases belong to this group. </li></ul>
  47. 66. 3.Hydrolases <ul><li>These enzymes catalyse cleavage reactions or the reverse fragment condensations.  </li></ul><ul><li>According to the type of bond cleaved, a distinction is made between peptidases, esterases, lipases, glycosidases, phosphatases and so on.  </li></ul><ul><li>Examples of this class of enzyme include; cholesterol esterase, alkaline phosphatase and glucoamylase. </li></ul>
  48. 67. 4.Lyases <ul><li>These enzymes non-hydrolytically remove groups from their substrates with the concomitant formation of double bonds or alternatively add new groups across double bonds.   </li></ul>
  49. 68. 5.Isomerases <ul><li>These enzymes catalyse intramolecular rearrangements and are subdivided into; </li></ul><ul><ul><ul><ul><ul><li>racemases </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>epimerases </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>mutases </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>cis - trans -isomerases </li></ul></ul></ul></ul></ul><ul><li>An example of this class of enzyme is glucose isomerase which catalyses the isomerisation of glucose to fructose. </li></ul>
  50. 69. 6.Ligases <ul><li>Ligases split C-C, C-O, C-N, C-S and C-halogen bonds without hydrolysis or oxidation.  </li></ul><ul><li>The reaction is usually accompanied by the consumption of a high energy compound such as ATP and other nucleoside triphosphates. </li></ul><ul><li>An example of this type of enzyme is pyruvate carboxylase which catalyses the following reaction: </li></ul><ul><li>pyruvate + HCO3- + ATP = Oxaloacetate + ADP + Pi </li></ul>
  51. 70. Formation of bonds with ATP cleavage   Ligases or Synthetases Isomerization reactions   Isomerases Addition to double bonds or its reverse   Lyases Hydrolysis reactions   Hydrolases Transfer of functional groups   Transferases   Oxidation-reduction reactions   Oxidases or Dehydrogenases   Type of Reaction Catalyzed   Group Name IEC Classification of Enzymes
  52. 71. <ul><li>Enzymes do NOT change the equilibrium position of the reaction, just the speed at which equilibrium is attained. </li></ul><ul><li>Most are globular or soluble. </li></ul><ul><li>Stereospecific (can recognize certain isomers only) due to the fact that amino acids of the active site are chiral themselves. </li></ul><ul><li>Substrate/s bind in hydrophobic cleft (active site) between several domains where catalysis occurs: </li></ul><ul><ul><li>Van der Waals forces </li></ul></ul><ul><ul><li>Hydrophobic interactions </li></ul></ul><ul><ul><li>Electrostatic interactions </li></ul></ul><ul><li>Active site has structure that is complimentary in structure to the structure of its substrate. </li></ul>PROPERTIES OF ENZYMES
  53. 72. <ul><li>Most are proteins, some are RNA. </li></ul><ul><li>Biological catalysts. </li></ul><ul><li>E + S  ES  EP  E + P </li></ul><ul><ul><li>Not changed by the reaction overall </li></ul></ul><ul><ul><li>Much higher reaction rates than uncatalyzed reactions. </li></ul></ul><ul><ul><li>Allow for biochemical reactions to occur under very mild conditions (temperature, near-neutral pH, 1 atm pressure) </li></ul></ul><ul><li>High yield of products (few side reactions or by-products) </li></ul><ul><li>Very specific reactions (specific for its substrate or a family of related substrates) </li></ul><ul><li>Often a regulated functions: </li></ul><ul><ul><li>allosteric activation or inhibition </li></ul></ul><ul><ul><li>covalent modification (phosphorylation changes) </li></ul></ul><ul><ul><li>enzyme expression controlled or cleavage of proenzyme controlled. </li></ul></ul>PROPERTIES OF ENZYMES
  54. 74. <ul><li>Describe what metabolism is? </li></ul><ul><li>What is the difference between anabolism and catabolism? </li></ul><ul><li>What is a substrate? </li></ul><ul><li>List 6 types of enzyme and state the characteristics each of them. </li></ul>EXERCISE: