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1. Enzymes By Khan Muhammad

2. Enzymes Enzymes are biological molecules that catalyze (i.e ., increase the rates of) chemical reactions . A catalyst is an agent which in minute quantity increases the velocity of chemical reactions of other substances without itself being destroyed or alter upon completion of reaction • In enzymatic reactions, substrates is converted into products. • Enzymes are selective for their substrates and speed up only a few reactions from among many possibilities.

3. Conti..

4. Characteristics lowering the activation energy. As a result, products are formed faster and reactions reach their equilibrium state more rapidly. • Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed reactions. • As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. • highly specific for their substrates. • A few RNA molecules called ribozymes also catalyze reactions

5. Conti.. • Inhibitors: decrease enzyme activity; • activators are molecules that increase activity. • Many drugs and poisons are enzyme inhibitors. • Activity is also affected by temperature, pressure, chemical environment (e.g., pH), and the concentration of substrate

6. Conti.. • Enzymes are in general globular proteins and range from just 62 amino acid residues in size, to over 2,500 residues • A small number of RNA-based biological catalysts exist, with the most common being the ribosome; these are referred to as either RNA-enzymes or ribozymes.

7. Conti.. only a small portion of the enzyme (around 2– 4 amino acids) is directly involved in catalysis. • The region that contains these catalytic residues, binds the substrate, and then carries out the reaction is known as the active site. • Enzymes can also contain sites that bind cofactors, which are needed for catalysis.

8. Conti.. Enzymes are long, linear chains of amino acids that fold to produce a three-dimensional product. • Individual protein chains may sometimes group together to form a protein complex. • Most enzymes can be denatured—that is, unfolded and inactivated—by heating or chemical denaturants, which disrupt the three-dimensional structure of the protein. • Depending on the enzyme, denaturation may be reversible or irreversible.

9. Lock and key model • Enzymes are very specific, • Nobel laureate organic chemist Emil Fischer in 1894 suggested • both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. • However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve.

10. Induced fit model In 1958, Daniel Koshland suggested a modification to the lock and key model: • Since enzymes are rather flexible structures, the active site is continuously reshaped by interactions with the substrate as the substrate interacts with the enzyme. • As a result, the substrate does not simply bind to a rigid active site; the amino acid side-chains that make up the active site are molded into the precise positions that enable the enzyme to perform its catalytic function. • In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site

11. Allosteric sites Allosteric sites are sites other than active sites on the enzyme that bind to molecules in the cellular environment. • The sites form weak, noncovalent bonds with these molecules , causing a change in the conformation of the enzyme. This change in conformation translates to the active site, which then affects the reaction rate of the enzyme. • Allosteric interactions can both inhibit and activate enzymes and are a common way that enzymes are controlled in the body.

12. Non protein components needed for enzymatic activity These includes derivatives of vitamins B and metallic ions. These enzymes are called holo enzymes (conjugated proteins). The protein part of such enzymes are called apoenzymes. and the non protein part are called prosthetic group which may, COFACTORS (if the prosthetic group is inorganic ions e.g. metals like iron, zinc and magnesium). COENZYMES (if the prosthetic group is an organic compounds e.g. usually derivatives of vitamins B complex like NAD, FAD and coenzymes-A, which are required by the enzymes for their activity.)

13. Classification of enzymes Enzymes may be classified in many different ways. In many cases their names end in the suffix ase which is preceded by the names of its substrates, e.g. sucrose, lipase, and urease etc. In other cases their names describes the action of an enzymes, e.g. transmethylase, oxidase etc. According to the enzyme commission (E.C) system, there are six main classes of enzymes Oxidoreductases Transferases Hydrolases Lyases Isomerases ligases

14. 1) Oxidoreductases These enzymes also called redox enzymes, catalyze oxidation – reduction reactions, and may further classified as. A) dehydrogenases It removes H 2 from the substrates If the reaction occurs in the presence of oxygen , it is called Aerobic dehydrogenase Glucose gluconolactones if it takes place in the absence of oxygen it is called anaerobic dehydrogenase Lactate pyruvate dehydrogenase Lactate dehydrogenase

15. B) Oxidases They use only oxygen as a hydrogen acceptor e.g. Ascorbic acid dehydroascorbate C) Oxygenases They incorporate oxygen into the substrate e.g. Phenylalanine tyrosine D) Hydrogen peroxidases There are two types i.e. peroxidases (found in plants) and catalases (found in animals). Both of them break down hydrogen peroxide (H 2 O 2 ) in different ways e.g. H 2 O 2 H 2 O + O 2 hydrolase Ascorbic oxidase catalase

16. 2) Transferases They catalyze the exchange of groups (except H2) between two compounds (from one substrate to another substrate). A) transaminase These enzyme catalyzes the exchange of amino group (-NH2) between an amino acid and keto acid, as a result amino acid becomes ketoacid and keto acid becomes amino acid e.g. ALT (alanine transaminase) and SGPT (serum glutamate pyruvate transaminase).

17. Conti.. ALT is an enzyme found in the liver that helps convert proteins into energy for the liver cells . When the liver is damaged, ALT is released into the bloodstream and levels increase. This test is sometimes referred to as SGPT . Aspartate transaminase (AST ) AST is an enzyme that helps the body break down amino acids.

18. B)phosphotransferases These enzymes catalyze the transfer of phosphate group also called kinases . glucose glucose 6- phosphate Hexokinase/ glucokinase ATP ADP

19. c) Transmethylase Catalyze the transfer of methyl group noradrenaline hippuric acid transmethylase S-adenosyl methionine(SAM) CH3

20. D) Transpeptidases These enzyme catalyze the transfer of peptide or amino acid s benzoic acid hippuric acid Transpeptidase Amino acid (glycine)

21. E) Transacylases Catalyzes the transfer of acyl group (S-CoA). E.g. Palmitic acid palmityl S-CoA transacylase CoA-SH

22. 3) hydrolases Catalyze hydrolysis (break done) of a compound by the addition of water into the substrate. Hydrolases are of different types depending upon the substrate Proteinases or proteases or proteolytic enzymes also called protein hydrolyzing enzymes, it may of two types. Exopeptidases. they act upon the terminal end of the polypeptide chains and are further divided into dipeptidases, tripeptidases and polypeptidases. Aminopolypeptidases, present in intestinal juice and acts upon the amino (-NH2) terminal of polypeptides. Carboxypolypeptidases, present in pancreatic juice , acts upon (-COOH),carboxyl terminal of polypeptides.

23. ii) endopeptidases. they act on the centrally located peptide bonds of the polypeptide chains. E.g. pepsin, present in gastric juice , trypsin and chymotrypsin, present in pancreatic juice.

24. B) Carbohydrate hydrolyzing enzymes (carbohydrases) These enzymes catalyze the hydrolysis of glycosidic bonds e.g. amylase, maltase, sucrase and lactase etc. Starch Maltase glucose amylase maltase

25. C) Lipid hydrolyzing enzymes They are of the following types: i) lipases As the name indicates, these enzymes break triglycerides (neutral fats) to release glycerol, fatty acids and mono or diglycerides. These enzymes is present in pancreatic juice and thus pancreatic juice is very important in fat digestion. ii) Cholesterases. They hydrolyze the cholesterol esters.

26. iii) Phospholipases (also called lecithinases) These are of four types i.e. A, B, C and D. Phospholipase –A is present in certain snake’s venom. It forms lysolecithin, which is a strong hemolyzing agent. D) Deaminases or Aminohydrolases. They remove –NH 2 group from an amino acid in the form of ammonia (NH 3 ), and convert that amino acid into corresponding keto acid e.g. Glutamic acid alpha-ketoglutarate deaminase H2O NH3

27. E) Deamidases or amidohydrolyases They remove an amide (HN) group from an amino acid in the form of ammonia (NH3) e.g. Asparagine aspartic acid Deamidases H2O NH3

28. F) Other hydrolyzing enzymes There are two group i.e. phosphatases and miscellaneous. phosphatases. they are of the following types phosphomonoestrases; split one phosphate group of a monoester. E.g. acid phosphatases and alkaline phosphatases. Phosphodiesterases; split off one phosphate group from diesters. Phosphorylase; add the phosphate group into the substrate. e.g. glycogen phosphorylase. Pyrophosphatases; remove pyrophosphate (two phosphates) from the substrate e.g. ATPase.

29. Nucleases or polynucleotidases; they decomposes nucleic acids (DNA and RNA). Nucleotidases; hydrolyze mononucleotides to nucleosides. a nucleotide consists of sugar, a nitrogenous base, and phosphate groups that number one to three. In contrast, a nucleoside involves a nitrogenous base that has a covalent attachment to sugar but with no phosphate group.

30. ii) Miscellaneous they are of the following types Cholinetrases; hydrolyze acetylcholine and other related substrate. Sulfatases; hydrolyze sulfate esters.

31. 4) Lyases They catalyze non-hydrolytic removal or addition (synthases) of groups at double bond They break the bond between a Carbon-Carbon Carbon-Oxygen Carbon-Sulfur The resultant molecule could have a double bond or ring For example: decarboxylases and aldolases Y B X A

32. Or Lyases can catalyze the breakdown (lysis) of a compound by removing a group from it, without the addition of water molecules (non hydrolytic), leaving a double bond A Y X B

33. X Y A B

34. X Y A B

35. a1 X Y A B

36. X Y A B

37. 5) Isomerases They catalyzes isomerization reactions Or they catalyze the formation of isomers of the substrate . X A Y B

38. A B X Y

39. 6) Ligases They catalyzes the synthesis of various (mostly C-X) bonds, where X may be oxygen, carbon, nitrogen and Sulphur. This is coupled with the breakdown of energy-containing substrates, usually ATP A P P P A B

40. 6. Ligases A B P A P P

41. Factors affecting enzyme activity Inhibitors: Species that decrease enzyme activity For example, some drugs and poisons Activators: Species that increases enzymatic activity Enzymatic activity is also affected by Temperature (High temperature denature proteins) Pressure Chemical environment (e.g., pH) Concentration of substrate Concentration of Enzyme

42. Substrate Concentration Increasing Substrate Concentration increases the rate of reaction This is because more substrate molecules will be colliding with enzyme However, after a certain concentration, any increase will have no effect on the rate Since Substrate Concentration will no longer be the limiting factor The enzymes will effectively become saturated They will be working at their maximum possible rate

43. Enzyme Concentration Increasing Enzyme Concentration will increase the rate of reaction As more enzymes will be colliding with substrate molecules However, this too will only have an effect up to a certain concentration Where the Enzyme Concentration is no longer the limiting factor

44. Temperature As temperature increases, initially the rate of reaction will increase d ue to increased Kinetic Energy However, at very high temperature the rate of reaction will begin to decrease, because it changes the enzyme native structure

45. PH Different enzymes have different Optimum pH values This is the pH value at which Active Site is the most Complementary to the shape of Substrate At the Optimum pH, the rate of reaction is at an optimum Any change in pH above or below they will quickly cause a decrease in the rate of reaction Since Active Sites shapes are not complementary to the shape of their Substrate Small changes do not cause permanent change to enzyme, since the bonds can be reformed Extreme changes in pH can cause enzymes to Denature and permanently lose their function

46. Enzyme Inhibition Inhibitor: A molecule that decreases the enzyme activity Inhibitors can cause two types of inhibition Reversible inhibition: the effect of an inhibitor can be reversed by decreasing the concentration of inhibitor Irreversible inhibition: there is no reversal of inhibition on decreasing the inhibitor concentration e.g. Cyanide: by covalently binding mitochondrial cytochrome oxidase Penicillin for bacterial peptidase

47. Types of Inhibitors Competitive Non-competitive Uncompetitive

48. Competitive inhibition Both the substrate and inhibitor compete for binding to the same active site of enzyme Enzyme can bind either substrate or Inhibitor not both at the same time If substrate binds, the ES complex is formed and then product is formed If inhibitor binds first then EI complex is formed and substrate cannot bind, so product is not formed The inhibition is most noticeable at low [S] but can be overcome at sufficiently high [S] S I E

49. Competitive inhibition Both the substrate and inhibitor compete for binding to the same active site of enzyme Enzyme can bind either substrate or Inhibitor not both at the same time If substrate binds, the ES complex is formed and then product is formed If inhibitor binds first then EI complex is formed and substrate cannot bind, so product is not formed The inhibition is most noticeable at low [S] but can be overcome at sufficiently high [S] I S E

50. Competitive inhibition Both the substrate and inhibitor compete for binding to the same active site of enzyme Enzyme can bind either substrate or Inhibitor not both at the same time If substrate binds, the ES complex is formed and then product is formed If inhibitor binds first then EI complex is formed and substrate cannot bind, so product is not formed The inhibition is most noticeable at low [S] but can be overcome at sufficiently high [S] S E P I

51. Competitive inhibition Both the substrate and inhibitor compete for binding to the same active site of enzyme Enzyme can bind either substrate or Inhibitor not both at the same time If substrate binds, the ES complex is formed and then product is formed If inhibitor binds first then EI complex is formed and substrate cannot bind, so product is not formed The inhibition is most noticeable at low [S] but can be overcome at sufficiently high [S] I S E

52. Competitive inhibition Both the substrate and inhibitor compete for binding to the same active site of enzyme Enzyme can bind either substrate or Inhibitor not both at the same time If substrate binds, the ES complex is formed and then product is formed If inhibitor binds first then EI complex is formed and substrate cannot bind, so product is not formed The inhibition is most noticeable at low [S] but can be overcome at sufficiently high [S] S I E

53. Competitive inhibition Both the substrate and inhibitor compete for binding to the same active site of enzyme Enzyme can bind either substrate or Inhibitor not both at the same time If substrate binds, the ES complex is formed and then product is formed If inhibitor binds first then EI complex is formed and substrate cannot bind, so product is not formed The inhibition is most noticeable at low [S] but can be overcome at sufficiently high [S] S I E X

54. Noncompetitive inhibition Inhibitor do not interfere in the binding of the substrate Inhibitor can bind both to the free enzyme or ES complex The product cannot be formed in both the cases However, if ES escapes inhibitor, then product is formed Increase in S cannot abolish the inhibition I S E

55. Noncompetitive inhibition Inhibitor do not interfere in the binding of the substrate Inhibitor can bind both to the free enzyme or ES complex The product cannot be formed in both the cases However, if ES escapes inhibitor, then product is formed Increase in S cannot abolish the inhibition I S E P

56. Noncompetitive inhibition Inhibitor do not interfere in the binding of the substrate Inhibitor can bind both to the free enzyme or ES complex The product cannot be formed in both the cases However, if ES escapes inhibitor, then product is formed Increase in S cannot abolish the inhibition I S E

57. Noncompetitive inhibition Inhibitor do not interfere in the binding of the substrate Inhibitor can bind both to the free enzyme or ES complex The product cannot be formed in both the cases However, if ES escapes inhibitor, then product is formed Increase in S cannot abolish the inhibition I S E

58. Noncompetitive inhibition Inhibitor do not interfere in the binding of the substrate Inhibitor can bind both to the free enzyme or ES complex The product cannot be formed in both the cases However, if ES escapes inhibitor, then product is formed Increase in S cannot abolish the inhibition I S E x

59. Noncompetitive inhibition Inhibitor do not interfere in the binding of the substrate Inhibitor can bind both to the free enzyme or ES complex The product cannot be formed in both the cases However, if ES escapes inhibitor, then product is formed Increase in S cannot abolish the inhibition I S E

60. Noncompetitive inhibition Inhibitor do not interfere in the binding of the substrate Inhibitor can bind both to the free enzyme or ES complex The product cannot be formed in both the cases However, if ES escapes inhibitor, then product is formed Increase in S cannot abolish the inhibition I S E

61. Noncompetitive inhibition Inhibitor do not interfere in the binding of the substrate Inhibitor can bind both to the free enzyme or ES complex The product cannot be formed in both the cases However, if ES escapes inhibitor, then product is formed Increase in S cannot abolish the inhibition I E S x

62. Uncompetitive inhibition Also known as anti-competitive inhibition It takes place when an enzyme is already bound to the substrate Inhibitor cannot bind the free enzyme Increasing S concentration cannot remove inhibition I S E

63. Uncompetitive inhibition Also known as anti-competitive inhibition It takes place when an enzyme is already bound to the substrate Inhibitor cannot bind the free enzyme Increasing S concentration cannot remove inhibition S I E

64. Uncompetitive inhibition Also known as anti-competitive inhibition It takes place when an enzyme is already bound to the substrate Inhibitor cannot bind the free enzyme Increasing S concentration cannot remove inhibition I S E

65. Uncompetitive inhibition Also known as anti-competitive inhibition It takes place when an enzyme is already bound to the substrate Inhibitor cannot bind the free enzyme Increasing S concentration cannot remove inhibition I S E

66. Uncompetitive inhibition Also known as anti-competitive inhibition It takes place when an enzyme is already bound to the substrate Inhibitor cannot bind the free enzyme Increasing S concentration cannot remove inhibition I S E

67. Uncompetitive inhibition Also known as anti-competitive inhibition It takes place when an enzyme is already bound to the substrate Inhibitor cannot bind the free enzyme Increasing S concentration cannot remove inhibition S E I x

68. Uncompetitive inhibition Also known as anti-competitive inhibition It takes place when an enzyme is already bound to the substrate Inhibitor cannot bind the free enzyme Increasing S concentration cannot remove inhibition S E I x

69. Properties of enzymes Enzymes are proteins in nature They never initiate any reaction instead they only accelerate the pre existing reactions They reduce the energy of activation.

70. Isozymes Isozymes: isoenzymes: Multiple forms of an enzyme They differ in amino acid sequence but catalyze the same chemical reaction Example: Lactate dehydrogenase (LDH)

71. Lactate dehydrogenase It is made by the combination of four subunits These subunits are of two different forms i.e. H-form and M-Form These subunits i.e. H and M, combine in different Combinations to make the tetramer These subunits can combine in the following 5 ways giving rise to 5 different isozymes Type Composition Location LDH1 HHHH Heart and Erythrocyte LDH2 HHHM Heart and Erythrocyte LDH3 HHMM Brain and Kidney LDH4 HMMM Skeletal Muscle and Liver LDH5 MMMM Skeletal Muscle and Liver

72. Zymogen: Proenzyme A zymogen: proenzyme: inactive enzyme precursor A zymogen requires a biochemical change such as a hydrolysis reaction change in the configuration It reveals the active site By which it becomes active

73. Example The pancreas secretes zymogens to prevent the enzymes from digesting proteins in the cells in which they are synthesised Enzymes like pepsin are created in the form of pepsinogen an inactive zymogen Pepsinogen is activated when Chief cells release it into HCl

74. Examples of zymogens Angiotensinogen Trypsinogen Chymotrypsinogen Pepsinogen Caspases Proelastase Prolipase Procarboxypolypeptidases

75. Cellular Regulation of Enzymes Reactions involved in a metabolic pathway are often grouped in sequences The first enzyme in such a pathway is usually a regulatory enzyme It controls the rate for the entire sequence A B C D F P The first enzyme is influenced by the concentration of the starting material [A] It may also be affected by the amount of final product [P] P inhibits E1 in a negative feedback manner E 2 E 1 E 3 E 4 E 5

76. Factors that can influence a regulatory enzyme The first enzyme in a pathway is regulated by the Concentration of the final product(s) Concentration of the beginning substrate Intermediates External factor like a hormone Combination of the above

77. Regulatory enzymes Actually enzymes activity can be regulated by two mechanisms. i.e. regulation of enzymes by non covalent modification (these enzymes are called allosteric enzymes), and the regulation of enzymes by covalent modification.

78. Regulation of enzyme by non covalent modification (allosteric enzymes) These enzymes have two or more subunits. In addition to the active site for their respective substrates, they also have a special site called allosteric sites. On these allosteric sites, reagents called Effectors can get bounded through non covalent bonds. This non covalent bonding of effectors may results in either an inhibition or an activation of the enzyme activity.

79. Allosteric inhibition Enzymes can be inhibited when an effector called negative effector or an allosteric inhibitor combine with the allosteric site of the enzyme. Feedback inhibition of enzymes Allosteric enzymes can be inhibited by the product of its own catalytic reaction. i.e. product in excess inhibition of the enzyme activity decreased product.

80. Regulation of enzymes by covalent modification Enzymes can be regulated by covalent modification. Regulation is most frequently done by the addition or removal of phosphate group from specific serine, threonine, or tyrosine residue of enzymes. addition of phosphate (phosphorylation) is catalyzed by a family of enzymes called protein kinases e.g. hexokinases, phosphofructokinases (glycolysis). This enzymes utilizes ATP as a phosphate donor.

81. Conti.. Removal of phosphate (dephosphorylation) from phosphorylated enzymes is catalyzed by a family of enzymes called phosphoprotein phosphatases e.g. glucose 6 phosphate phosphatase (gluconeogenesis). Addition or removal of phosphate group to and from an enzyme will result either in activation or inhibition of the enzymes.

82. Enzymes in clinical diagnosis Alkaline phosphatas ; marker of Paget's disease (Bone deformation), rickets (Bone softening due to vit D lack), cholestasis (bile flow slow/blocked), cirrhosis (scarring of the liver and poor liver function) , bone tumours, Pregnancy and childhood Acid phosphatase; Marker of prostate cancer and tumor Aspartate amino transferase; marker of myocardial infarction and liver diseases. Alanine amino transferase; very high values in acute hepatitis and Moderate increase may be seen in chronic liver disease lactate dehydrogenase ; is a marker for hemolytic anemias, (Red blood cell destroys), hepatocellular damage, muscular dystrophy, (muscle weakness and loss), carcinomas, (cancers), leukemias, increase of immature wbcs (blood cancer) Creatine kinase ; increases in polymyositis (muscle disease), Duchenne muscular dystrophy (muscle weakness), myocardial infarction, following surgery and severe exercise Nucleotide phosphatase; It is moderately increased in hepatitis and highly elevated in biliary obstruction.

83. Conti.. Amylase; splits starch to maltose. It is activated by calcium, chloride and fluoride ions. It is produced by pancreas and salivary glands. Its value is increased about 1000 times in acute pancreatitis Lipase; present in pancreatic secretion, elevated in acute pancreatitis Aldolase; tetrameric glycolytic enzyme with 5 iso -enzymes. elevated in muscle damages Enolase; a glycolytic enzyme. tumor marker for cancers Cholinesterase; present in nerve endings and in RBCs. Plasma cholinesterase activity falls in organophosphate poisoning. Gamma glutamyl transferase; Value increases in infective hepatitis and prostate cancers. alcoholism, obstructive jaundice

84. Conti.. Serum glutamate pyruvate transaminase (SGPT) or Alanine Transaminase (ALT).