Kuliah biokimia enzim

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  • This example demonstrates how an end product can inhibit the first step in its production. Isoleucine binds to the allosteric site of threonine deaminase and prevents threonine from binding to the active site because the shape of the active site is altered. When the level of isoleucine drops in the cell’s cytoplasm, the isoleucine is removed from the allosteric site on the enzyme, the active site resumes the activated shape and the pathway is “cut back on” and isoleucine begins to be produced.
  • Kuliah biokimia enzim

    1. 1. Enzymes Compiled by : dr. Santoso
    2. 2. <ul><li>Introduction </li></ul><ul><li>Mechanisms of Enzymatic reaction </li></ul><ul><li>Kinetic of enzyme activity </li></ul><ul><li>Factor affecting enzyme activity </li></ul><ul><li>Regulation of enzyme activity </li></ul>
    3. 3. <ul><li>Introduction </li></ul><ul><li>Mechanisms of Enzymatic reaction </li></ul><ul><li>Kinetic of enzyme activity </li></ul><ul><li>Factor affecting enzyme activity </li></ul><ul><li>Regulation of enzyme activity </li></ul>
    4. 4. What Are Enzymes? <ul><li>Most enzymes are Proteins ( tertiary and quaternary structures) </li></ul><ul><li>Act as Catalyst to accelerates a reaction </li></ul><ul><li>Not permanently changed in the process </li></ul>
    5. 5. Enzymes <ul><li>Are specific for what they will catalyze </li></ul><ul><li>Are Reusable </li></ul><ul><li>End in – ase </li></ul><ul><li>-Sucrase </li></ul><ul><li>-Lactase </li></ul><ul><li>-Maltase </li></ul>
    6. 6. <ul><li>Introduction </li></ul><ul><li>Mechanisms of Enzymatic reaction </li></ul><ul><li>Kinetic of enzyme activity </li></ul><ul><li>Factor affecting enzyme activity </li></ul><ul><li>Regulation of enzyme activity </li></ul>
    7. 7. How do enzymes Work? Enzymes work by weakening bonds which lowers activation energy
    8. 8.
    9. 9. How do Enzymes Affect Reaction Rates ? <ul><li>Enzymes affect the rates of reactions by lowering the amount of energy of activation required for the reactions to begin. Therefore processes can occur in living systems at lower temperatures or energy levels than it would require for these same reactions to occur without the enzymes present. </li></ul>
    10. 10. How do Enzymes Bind to Substrates <ul><li>There are two proposed methods by which enzymes bind to their substrate molecules: </li></ul><ul><ul><li>Lock and Key Model </li></ul></ul><ul><ul><li>Induced-Fit Model </li></ul></ul>
    11. 11. Enzyme-Substrate Complex The substance (reactant) an enzyme acts on is the substrate Substrate Joins Enzyme
    12. 12. Active Site <ul><li>A restricted region of an enzyme molecule which binds to the substrate . </li></ul>Substrate Active Site Enzyme
    13. 13. Lock and Key Model Enzyme returns from the reaction unchanged and can now react with more substrate. enzyme S1 S2 S2 enzyme S1 ENZYME SUBSTRATE COMPLEX enzyme SUBSTRATE MOLECULES Active site P P Products
    14. 14. Induced Fit <ul><li>A change in the shape of an enzyme’s active site </li></ul><ul><li>Induced by the substrate </li></ul>
    15. 15. Induced Fit <ul><li>A change in the configuration of an enzyme’s active site (H+ and ionic bonds are involved). </li></ul><ul><li>Induced by the substrate . </li></ul>Enzyme Active Site substrate induced fit
    16. 16. Induced-Fit Model
    17. 17. Enzyme Cooperativity <ul><li>Some enzymes have multiple active site. It has been observed that when one substrate molecule binds to a single active site in the inactive form or tense state of the enzyme, a configurational change occurs in the other active sites making them more receptive to other substrate molecules. </li></ul>
    18. 18. <ul><li>Introduction </li></ul><ul><li>Mechanisms of Enzymatic reaction </li></ul><ul><li>Kinetic of enzyme activity </li></ul><ul><li>Factor affecting enzyme activity </li></ul><ul><li>Regulation of enzyme activity </li></ul>
    19. 19. Enzyme Kinetics <ul><li>Expression for enzyme catalyzed reaction: </li></ul>
    20. 20. Michaelis-Menten Equation <ul><li>Rate increase with [S] </li></ul><ul><li>Rate levels off as approach V max </li></ul><ul><ul><li>More S than active sites in E </li></ul></ul><ul><ul><li>Adding S has no effect </li></ul></ul><ul><li>At V 0 = ½ V max </li></ul><ul><ul><li>[S] = K M </li></ul></ul>V 0 = V max [S] / K M + [S]
    21. 21. <ul><li>V max occurs when enzyme active sites are saturated with substrate </li></ul><ul><li>K m (Michaelis-Menten constant) reflects affinity of enzyme for its substrate </li></ul><ul><li>smaller the K m , the greater the affinity an enzyme has for its substrate </li></ul>
    22. 22. <ul><li>Introduction </li></ul><ul><li>Mechanisms of Enzymatic reaction </li></ul><ul><li>Kinetic of enzyme activity </li></ul><ul><li>Factor affecting enzyme activity </li></ul><ul><li>Regulation of enzyme activity </li></ul>
    23. 23. What Affects Enzyme Activity? <ul><li>Three factors: </li></ul><ul><li>Environmental Conditions </li></ul><ul><li>Cofactors and Coenzymes </li></ul><ul><li>Enzyme Inhibitors </li></ul>
    24. 24. 1. Environmental Conditions <ul><li>Extreme Temperature are the most dangerous </li></ul><ul><ul><li>high temps may denature (unfold) the enzyme. </li></ul></ul><ul><li>pH (most like 6 - 8 pH near neutral) </li></ul><ul><li>Ionic concentration (salt ions) </li></ul>
    25. 25. Temperature <ul><li>All enzymes have an optimum temperature at which they work best. If you observe the enzyme’s activity below the specific temperature it will steadily increase until it reaches the optimum. After the optimum temperature is reached the enzymes activity drops dramatically due to denaturing. </li></ul>Depending on the species, the range of optimum activity is very broad. Above is a comparison of human enzyme activity with that of bacteria found in hot springs and oceanic vents.
    26. 26. pH <ul><li>All enzymes have an optimum pH at which they work best. If the pH falls below or rises above the optimum value, enzymatic activity decreases </li></ul><ul><li>as a result of denaturing. </li></ul>In the human body’s digestive tract there are variations in pH from area to area. The stomach’s juices’ pH is around 2 (acidic), the enzyme pepsin found in the gastric juices has optimum activity at a pH of 2. The small intestine’s juice’s pH is around 8 (basic). The enzyme trypsin found in the small intestine’s juices has optimum activity at a pH of 8.
    27. 27. Substrate Concentration <ul><li>The concentration of substrate also has an affect on the rate of enzyme activity. If the concentration of substrate is increased while the concentration of enzyme is constant, the level of enzyme activity will increase until a point of saturation is reached. At this point there are no enzymes available to react with excess substrate and the rate of the reaction stabilizes. No matter if you continue to add substrate, the reaction rate will not increase! </li></ul>Increasing Substrate Concentration Rate of Reaction Point of Saturation, all active sites are filled with substrate.
    28. 28. 2. Cofactors and Coenzymes <ul><li>Inorganic substances (zinc, iron) and vitamins (respectively) are sometimes need for proper enzymatic activity . </li></ul><ul><li>Example: </li></ul><ul><li>Iron must be present in the quaternary structure - hemoglobin in order for it to pick up oxygen. </li></ul>
    29. 29. <ul><li>Coenzymes are bound at the active site in order to interact with the substrate and play an essential role in the catalysed reaction. </li></ul><ul><li>They act as carriers of a variety of chemical groups. </li></ul>
    30. 30. Most water-soluble vitamins are components of coenzymes Vitamin Coenzyme Deficiency Thiamine (B 1 ) Thiamine pyrophosphate Beriberi (weight loss,other problems Riboflavin (B 2 ) FAD + Mouth lesions, dermatitis Nicotinic acid (niacine) NAD + Pellagra (dermatitis, depression) Pantohtinic acid Coenzyme A Hypertension Biotin Biotin Rash, muscle pain
    31. 31. 3. Enzyme Inhibitors <ul><li>Specific for an enzyme </li></ul><ul><li>Can be reversible or non-reversible </li></ul><ul><li>Competitive inhibitors </li></ul><ul><li>Non-competitive inhibitors </li></ul>
    32. 32. Competitive inhibitors chemicals that resemble an enzyme’s normal substrate and compete with it for the active site . Substrate Enzyme Competitive inhibitor
    33. 33. Noncompetitive Inhibitors Inhibitors that do not enter the active site , but bind to another part of the enzyme causing the enzyme to change its shape , which in turn alters the active site . Substrate Enzyme active site altered Noncompetitive Inhibitor
    34. 34. Competitive vs. Non-competitive inhibitors
    35. 35. <ul><li>Introduction </li></ul><ul><li>Mechanisms of Enzymatic reaction </li></ul><ul><li>Kinetic of enzyme activity </li></ul><ul><li>Factor affecting enzyme activity </li></ul><ul><li>Regulation of enzyme activity </li></ul>
    36. 36. Enzyme activity is regulated by four different mechanisms* <ul><li>(1) Allosteric control </li></ul><ul><li>(2) Covalent modification </li></ul><ul><li>(3) Proteolytic activation </li></ul><ul><li>(4) Stimulation and inhibition by control proteins </li></ul><ul><li>* changes in enzyme levels due to regulation of protein synthesis or degradation are additional, long-term ways to regulate enzyme activity </li></ul>
    37. 37. Allosteric regulation of enzyme activity <ul><li>Allosteric regulation = the activation or inhibition of an enzyme’s activity due to binding of an effector molecule at a regulatory site that is distinct from the active site of the enzyme </li></ul><ul><li>Allosteric regulators generally act by increasing or decreasing the enzyme’s affinity for the substrate </li></ul>
    38. 38. Allosteric regulation Many allosterically controlled enzymse show quaternary structure
    39. 39. Covalent modification regulates the catalytic activity of some enzymes <ul><li>Can either activate it or inhibit it by altering the conformation of the enzyme or by serving as a functional group in the active site. </li></ul>
    40. 40. Biotin <ul><li>Biotin serves as a CO 2 carrier and is essential for pyruvate carboxylase, participates directly in the catalytic mechanism of the enzyme (as opposed to inducing a conformational change in the enzyme that indirectly affects the activity of the enzyme). </li></ul>
    41. 41. Phosphorylation - an example of regulation by reversible covalent modification of the enzyme <ul><li>Inserting a negatively charged phosphate group into the appropriate location in an enzyme can induce a conformational change in the enzyme that either increases, or decreases, its activity. </li></ul>
    42. 42. Top 5 reasons why phosphorylation is used to regulate enzyme activity: <ul><li>Phosphorylation is rapidly reversible , making it possible to quickly switch between active and inactive forms of an enzyme. </li></ul><ul><li>Phosphorylation is relatively inexpensive since it does not require the synthesis of new protein molecules. </li></ul><ul><li>Results in large ∆ G rxn for the phosphorylation reaction. Phosphorylation can shift the conformational equilibrium of a protein by a factor of ≈10 4 . </li></ul><ul><li>Phosphorylation/dephosphorylation is rapid and its timing can be adjusted to meet the physiological needs of the cell. </li></ul><ul><li>Phosphorylation effects can be rapidly amplified via a kinase cascade. </li></ul>
    43. 43. Summary: Covalent modification <ul><li>Covalent modification allows an enzyme to be rapidly activated or inactivated </li></ul><ul><li>With covalent modification, regulation of a enzyme activity is achieved at low energy costs to the cell (i.e. regulation does not require synthesis of a new enzyme or inhibitory protein). </li></ul><ul><li>Phosphorylation is a good example of how enzymes are activated and inactivated by covalent post-translational modifications </li></ul>
    44. 44. <ul><li>Proteolytic activation </li></ul>
    45. 45. <ul><li>Such as those involved in protein digestion, blood clotting, and bone and tissue remodeling, must be kept in a completely inactive state until they are needed. These enzymes are synthesized as inactive precursors (known as zymogens or proenzymes) and activated when needed by proteolytic cleavage of a specific peptide bond in the zymogen. </li></ul>Proteolytic activation
    46. 46. Regulation of digestive enzymes
    47. 47. <ul><li>Digestion of proteins requires simultaneous activation of several digestive enzymes. </li></ul><ul><li>This is achieved by synthesizing the digestive enzymes as inactive zymogens that are activated by specific proteolysis by trypsin. </li></ul><ul><li>Trypsin is activated by enteropeptidase catalyzed proteolysis of a unique lysine-isoleucine peptide bond (this is the “master switch” that turns on the activation of the digestive enzymes). </li></ul>
    48. 48. Pepsinogen is converted to pepsin by autocatalytic proteolysis at pH 2 <ul><li>Pepsinogen has a low amount of activity at pH 2, allowing it to cleave the peptide bind between amino acids 43 and 44 to generate pepsin, that is much more active than pepsinogen. </li></ul>
    49. 49. Zymogen Pepsinogen Chymotrypsinogen Trypsinogen Procarboxypeptidase Proelastase Prothrombin Fibrinogen Factor VII Factor X Proinsulin Procollagen Procollagenase Active Enzyme Pepsin Chymotrypsin Trypsin Carboxypeptidase Elastase Thrombin Fibrin Factor VIIa Factor Xa Insulin Collagen Collagenase Function protein digestion protein digestion protein digestion protein digestion protein digestion blood clot formation blood clot formation blood clot formation blood clot formation plasma glucose homeostasis component of skin and bone remodeling processes during metamorphosis, etc.
    50. 50. <ul><li>Digestive enzymes, blood clotting enzymes, and enzymes involved in bone and tissue remodeling catalyze reactions that would be disastrous if they occurred at inappropriate times or locations. </li></ul><ul><li>For example, if proteolytic digestion of proteins occurred in the pancreas, they would start digesting the pancreas itself. Similarly, if blood clotting factors are activated when they aren’t needed, they will initiate blood clotting throughout the body. </li></ul><ul><li>So, they are synthesized as inactive zymogens and are stored in this inactive state until they are needed. </li></ul>
    51. 51. Blood clot formation - an example of zymogen activations
    52. 52. <ul><li>Blood clotting is an excellent example of a proteolytic cascade designed to amplify an external signal (e.g. trauma) and evoke a rapid response (blood clot formation). </li></ul><ul><li>Thrombin itself is inhibited by antithrombin (a serpin). This provides the body with a mechanism to prevent random blood clot formation beyond the site of injury. </li></ul>
    53. 53. <ul><li>It can occur outside of cells, since ATP is not needed to convert the zymogen into the active form of the enzyme. </li></ul><ul><li>It is not a reversible reaction. Inactivation of the active enzyme must occur by either degradation of the enzyme or by inhibition (e.g. due to the binding of an inhibitory protein to the active enzyme). </li></ul>Proteolytic cleavage differs from phosphorylation
    54. 54. Stimulation and inhibition by control proteins <ul><li>Some enzymes have regulatory proteins that bind to them and regulate their activity. cAMP-dependent protein kinase is one examples of this type of regulation. </li></ul>
    55. 55. Serpins - An example of inhibition by control proteins <ul><li>Once trypsin is activated, we need a mechanism to turn it off when it is no longer needed. Pancreatic trypsin inhibitor is used to shut off trypsin activity </li></ul><ul><ul><li>pancreatic trypsin inhibitor binds very tightly to trypsin </li></ul></ul><ul><ul><li>pancreatic trypsin inhibitor is a member of a class of proteins known as ser ine p rotease in hibitors ( serpins ). </li></ul></ul><ul><ul><li>Serpins are polypeptides that inhibit serine proteases by binding to the active sites of these enzymes. </li></ul></ul>Trypsin (orange) bound to bovine pancreatic trypsin inhibitor (violet). His 57, Asp 102, Gly 193, and Ser 195 in the active site of trypsin are shown in green, red, cyan, and blue, respectively. Lys 15 in BPTI forms a salt bridge with Asp 189 in trypsin in the trypsin:BPTI complex. Binding of bovine pancreatic trypsin inhibitor is essentially irreversible .
    56. 56. Elastase is inhibited by α 1-antitrypsin <ul><li>α 1-antitrypsin inhibits elastase, a serine protease that is responsible for remodeling collagen. </li></ul><ul><li>Individuals in which Glu 342 in α 1-antitrypsin is replaced by a lysine secrete only 15% of the normal levels for α 1-antitrypsin, resulting in uncontrolled elastase activity and the breakdown of the alveolar walls in the lung. </li></ul>
    57. 57. <ul><li>Note that although serpins are tight binding inhibitors or serine proteases, they do not form covalent bonds with the serine proteases. </li></ul><ul><li>In other words, binding of serpins to serine proteases does not involve the formation of covalent bonds between the serpins and the serine proteases. </li></ul>
    58. 58. Summary of regulatory mechanisms <ul><li>Allosteric regulation </li></ul><ul><ul><li>ATP activation/CTP inhibition of ATCase sigmoidal kinetics </li></ul></ul><ul><ul><li>cAMP activation of cAMP-dependent protein kinase </li></ul></ul><ul><li>Reversible covalent modification </li></ul><ul><ul><li>Phosphorylation </li></ul></ul><ul><ul><li>Ser/Thr protein kinases, Tyr kinases, kinase cascades </li></ul></ul><ul><li>Proteolytic activation </li></ul><ul><ul><li>Digestive enzyme, blood clotting factors </li></ul></ul><ul><li>Protein activators and inhibitors </li></ul><ul><ul><li>Serpins </li></ul></ul>
    59. 59. Regulating the rates of enzyme-driven reactions <ul><li>Cells use inhibitors and activators to turn off and on enzymes </li></ul><ul><li>Many enzymes are controlled by an allosteric site remote from the active site </li></ul>
    60. 60. Enzyme 1 Enzyme 2 Enzyme 3 Inter- mediate Inter- mediate X Product Start of pathway Presence of product inhibits enzyme 1 Feedback inhibition Many enzymes are actually regulated by the end products of the reaction they catalyze This prevents too much product from being made
    61. 61. An example of Feedback inhibition This example demonstrates how an end product can inhibit the first step in its production. Isoleucine binds to the allosteric site of threonine deaminase and prevents threonine from binding to the active site because the shape of the active site is altered. When the level of isoleucine drops in the cell’s cytoplasm, the isoleucine is removed from the allosteric site on the enzyme, the active site resumes the activated shape and the pathway is “cut back on” and isoleucine begins to be produced.
    62. 62. <ul><li>THANK YOU </li></ul>

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