Lec 4 level 3-nu (enzymes)


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Lec 4 level 3-nu (enzymes)

  1. 1. Nursing - 4Enzymes
  2. 2. • Enzymes• Are protein catalysts that increase the rate of reactions without being change in the overall process.• All reactions in the body are mediated by enzymes.2
  3. 3. Nomenclature of enzymes• Each enzyme is assigned two names.• The first is its short, recommended name, convenient for every day use.• The second is the more complete systematic name, which is used when an enzyme must be identified without ambiguity. 3
  4. 4. A.Recommended name:• Most commonly used enzyme names have the suffix “-ase” attached to the substrate of the reaction (e.g. urease),• Or to description of the action performed (e.g. lactate dehydrogenase).• Note: Some enzymes retain their original trivial names, which give no hint of the associated enzymatic reaction, e.g. trypsin and pepsin. 4
  5. 5. B. Systematic name:• In the systematic naming system, enzymes are divided into six major classes, each with numerous subgroups.• For a give 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+ oxi-dehydrogenase.• Note: Each enzyme is also assigned a classification number.• The systematic names are informative, but are frequently too cumbersome to be general use. 5
  6. 6. Classification of enzymesAccording to its function• Class 1. Oxidoreductase:Transfer of hydrogen, catalyze oxidation-reduction reaction as conversion of lactate to pyruvate by lactate dehydrogenase.• Class 2. Transferase:Catalyze transfer of C-, N-, or P- containing groups. Such as conversion of serine to glycine by serine hydroxy methyl transferase• Class 3. Hydrolases:Catalyze cleavage of bonds by addition of water, e.g. Urease convert urea to CO2 and NH3 6
  7. 7. • Class 4. Lyases:Cleave without adding water, catalyze cleavage of C-C, C-S and certain C-N bonds, such as cleavage of pyruvate by pyruvate decarboxylase into acetaldehyde.• Class 5. Isomerases:Catalyze racemization of optical or geometric isomers, such as conversion of methylmalonyl CoA to Succinyl CoA by methylmalonyl CoA mutase. 7
  8. 8. • Class 6. Ligases:ATP dependent condensation of twomolecules, e.g. acetyl CoA carboxylase. Itcatalyze formation of bonds betweencarbon and O, S, N coupled to hydrolysisof high-energy phosphatases, such asconversion of pyruvate to oxaloacetate bypyruvate carboxylase.8
  9. 9. Properties of Enzymes• 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. 9
  10. 10. A.Active sites:• Enzyme molecules contain a special pocket or cleft called the active site.• The active site contains amino acid chains 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 enzyme (induced fit) that allows catalysis.• ES is converted to an enzyme-product (EP) complex that subsequently dissociates to enzyme and product. 10
  11. 11. Active Site11
  12. 12. 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. 12
  13. 13. 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. 13
  14. 14. D. Holoenzymes:• Some enzymes require molecules other than proteins for enzymatic activity.• The term holoenzyme refers to the active enzyme with its nonprotein component.• The term apoenzyme is inactive enzyme without its nonprotein part.• If the nonprotein part is a metal ion such as Zn 2+ or Fe2+, it is called a cofactor.• If it is a small organic molecule, it is termed a coenzyme. 14
  15. 15. • Coenzymes that only transiently associate with the enzyme are called co-substrates.• If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group as FAD.• Coenzymes frequently are derived from vitamins.• Example, NAD+ contains niacin and FAD contains riboflavin. 15
  16. 16. 16
  17. 17. E. Regulation:Enzyme activity can be regulated, that is, increasedor decreased, so that the rate of product formationresponds to cellular need.F. Location within the cell:• Many enzymes are localized in specific organelles within the cell.• Such compartmentalization serves to isolate the reaction substrate or product from other competing reactions. 17
  18. 18. How Enzymes Work• The mechanism of enzyme action can be viewed from two different perspectives.• The first treats catalysis in terms of energy changes• The second perspective describe how the active site chemically facilitates catalysis. 18
  19. 19. B. Chemistry of the active siteActive site is a complex molecule thatfacilitate the conversion of substrate to product. 19
  20. 20. 20
  21. 21. Factors influencing enzyme activity1- Enzyme concentration:• Velocity of reaction is increased proportionately with the concentration of enzyme, when substrate concentration is unlimited.2- Substrate concentration:• As substrate concentration is increased, the velocity is also correspondingly increased in the initial phases; but the curve flattens afterwards. The maximum velocity thus obtained is called Vmax.3- Effect of concentration of products:• When product concentration is increased, the reaction is slowed, stopped or even reversed. 21
  22. 22. Substrate concentration 22
  23. 23. 4. Temperature23
  24. 24. Effect of temperature:• The velocity of enzyme reaction increases when temperature of the medium is increased; reaches a maximum and then falls.• As temperature is increased, more molecules get activation energy, or molecules are at increased rate of motion. So their collision probabilities are increased and so the reaction velocity is enhanced.• But when temperature is more than 50°C, heat denaturation and consequent loss of tertiary structure of protein occurs. So activity of the enzyme decreased.• Most human enzymes have the optimum temperature around 37°C. Certain bacteria living in hot springs will 24 have enzymes with optimum temperature near 100°C.
  25. 25. 5. Effect of pH• Each enzyme has an optimum pH.• Usually enzymes have the optimum pH between 6 and 8.• Some important exceptions are Pepsin (with optimum pH 1-2), alkaline phosphatase (optimum pH 9-10) and acid phosphatase (4-5). 25
  26. 26. Michaelis-Menten EquationA. Reaction model:• This also called enzyme-substrate complex theory.• The enzyme (E) combines with the substrate (S), to form an enzyme-substrate (ES) complex, which immediately breaks down to the enzyme and the product (P). k1 k2• E + S ↔ E-S complex → E + P k-1Where, K1, k-1 and k2 are rate constants. 26
  27. 27. 27
  28. 28. B. Michaelis-Menten Equation: Vmax [S] V0 = -------------------- Km + [S]Where,• V0 = initial reaction velocity• Vmax = maximal velocity• Km = Michaelis = (k-1 + k2)/k1• [S] = substrate concentration 28
  29. 29. Inhibition of enzyme activity• Inhibitor is any substance that can diminish the velocity of an enzyme-catalyzed reaction.• Types of inhibitors:1. Irriversible inhibitors bind to enzyme throughcovalent bonds.2. Reversible inhibitors typically bind to enzymethrough noncovalent bonds, thus dilution of theenzyme-inhibitor complex results in dissociation ofthe reversibly bound inhibitor, and recovery of enzymeactivity.• Reversible inhibition are competitive and noncompetitive. 29
  30. 30. A. Competitive inhibition:• In this type, the inhibitor there will be similarity in three-dimensional structure between substrate (S) and inhibitor (I).• The inhibitor molecules are competing with the normal substrate molecules for attaching with the active site of the enzyme.• E + S → E-S → E + P• 30 E + I → E-I
  31. 31. 31
  32. 32. 32
  33. 33. • If substrate concentration is high when compared to inhibitor, then the inhibition is reversed.• For example, the succinate dehydrogenase reaction is inhibited by malonate, which are structural analogs of succinate. 33
  34. 34. B- Noncompetitive inhibition• A variety of poisons, such as iodoacetate, heavy metal ions (silver, mercury) and oxidizing agents act as irreversible noncompetitive inhibitors.• The inhibitor usually binds to different domain on the enzyme, other than the substrate binding site.• Since these inhibitors have no structural resemblance to the substrate, an increase in the substrate concentration generally does not relieve this inhibition. 34
  35. 35. 35
  36. 36. • Cyanide inhibits cytochrome oxidase. Fluoride will remove magnesium ions and will inhibit the enzyme, enolase, and consequently the glycolysis.• The velocity of the reaction is reduced.• Increasing substrate concentration will not abolish non-competitive inhibition. 36
  37. 37. 37
  38. 38. Enzyme Inhibitors as Drugs• The widely prescribed β-lactan antibiotics, such as penicillin and amoxicillin, act by inhibiting enzymes involved in bacterial cell wall synthesis. 38
  39. 39. Allosteric enzyme:• Allosteric enzyme has one catalytic site where the substrate binds and another separate allosteric site where the modifier binds (allo=other).• Allosteric enzymes are utilized by the body for regulating metabolic pathways. Such a regulatory enzyme in a particular pathway is called the key enzyme or rate limiting enzyme. 39
  40. 40. 40
  41. 41. 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. 41
  42. 42. • 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. 42
  43. 43. A. Alteration of plasma enzyme levelsin disease states• Many diseases that cause tissue damage result in an increased release of intracellular enzymes into the plasma.• The activities of many of these enzymes are routinely determined for diagnostic purposes in diseases of the heart, liver, skeletal muscle, and other tissues.• The level of specific enzyme activity in the plasma frequently correlates with the extent of tissue damage.• Thus, determining the degree of elevation of a particular enzyme activity in the plasma is often useful in evaluating the prognosis for the patient. 43
  44. 44. B. Plasma enzymes as diagnostic tools:• Some enzymes show relatively high activity in only one or a few tissues.• The presence of increased levels of these enzymes in plasma thus reflects damage to the corresponding tissue.• For example, the enzyme alanine aminotransferase (ALT) is abundant in the liver.• The appearance of elevated levels of ALT in plasma signals possible damage to hepatic tissue.• Note: Measurement of ALT is part of the liver function test panel.• Increases in plasma levels of enzymes with a wide tissue distribution provide a less specific indication of the site of cellular injury and limits their diagnostic value. 44
  45. 45. C. Isoenzymes and diseases of the heart:• Most isoenzymes (also called isozymes) are enzymes that catalyze the same reaction.• However, they do not necessarily have the same physical properties because of genetically determined differences in amino acid sequence.• For this reason, isoenzymes may contain different numbers of charged amino acids and may, therefore, be separated from each other by electrophoresis. 45
  46. 46. • Different organs frequently contain characteristic properties of different isoenzymes.• The pattern of isoenzymes found in the plasma may, therefore, serve as a means of identifying the site of tissue damage.• For example, the plasma levels of creatine kinase (CK) are commonly determined in the diagnosis of myocardial infarction.• They are particularly useful when the electrocardiogram is difficult to interpret, such as when there have been previous episodes of heart disease. 46
  47. 47. 1. Quaternary structure of isoenzymes• Many isoenzymes contain different subunits in various combinations.• For example, creatine kinase (CK) occurs as three isoenzymes.• Each isoenzyme is a dimer composed of two polypeptides (called B and M subunits) associated in one of three combinations: CK1 = BB, CK2 = MB, and CK3 = MM.• Each CK isoenzyme shows a characteristic electrophoretic mobility.• Note: Virtually all CK in the brain is the BB isoform, whereas in skeletal muscle it is MM. In cardiac muscle, about one-third is MB with the rest as MM 47
  48. 48. 2. Diagnosis of myocardial infarction• Measurement of blood levels of proteins with cardiac specificity is used in diagnosis of myocardial infarction (MI) because myocardial muscle is the only tissue that contains more than 5% of the total CK activity as the CK2 (MB) isoenzyme.• The most sensitive and earlier marker of acute myocardial infarction (AM) is either Troponin I or Troponin T. 48