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Enzymes /certified fixed orthodontic courses by Indian dental academy

  1. 1. INDIAN DENTAL ACADEMY Leader in continuing dental education www.indiandentalacademy.com www.indiandentalacademy.com
  2. 2. CONTENTS 1. What are enzymes? 2. Definitions & Exception 3. History 4. Enzymes in Everyday life 5. Food enzyme concept 6. Mode of action 7. Enzymes in relation to ΔG & law of conservation of energy 8. Coenzymes 9. Metallo enzymes 10. Active site of enzyme www.indiandentalacademy.com
  3. 3. 11. Major classes 12. Theories 13. Enzyme kinetics 14. Factors affecting enzyme activity 15. Enzyme inhibition 16. Substrate specificity 17. Regulation of enzyme activity 18. Mixed inhibition 19. Bisubstrate reaction 20. Catalytic Mechanism 21. LYSOZYME : A model of enzyme action 22. Restriction enzyme 23. Clinical / Pathological importance of enzymes 24. Enzyme therapy Vs Cancer 25. Enzyme detection www.indiandentalacademy.com
  4. 4. WHAT ARE ENZYMES? www.indiandentalacademy.com
  5. 5. DEFINITION Enzymes are biological substances which are proteins that act as catalysts and help complex reaction occur everywhere in life. OR Garden defines enzymes as any of numerous complex proteins that are produced by living cells facilitating naturally occurring biochemical reactions at body temperature. OR Enzymes or proteins which act as catalysts in various biochemical reactions of our body by lowering activation energy. www.indiandentalacademy.com
  6. 6. EXCEPTION Ribozymes : these are made of RNA instead of proteins catalyzing RNA splicing. www.indiandentalacademy.com
  7. 7. HISTORY “Word enzyme” comes from greek word “in Leaven”. The name enzyme was coined by Fredrich Willhelm Kuhne in 1878. Early history of enzymology, study of enzymes is largely together that of from biochemistry, nineteenth these disciplines century evolved investigations of Fermentation and digestion. Research on fermentation began in 1810 with Joseph Lussac’s determination that ethanol and CO2 are principle products of sugar decomposition by yeast. www.indiandentalacademy.com
  8. 8. In 1835 Jacob Brazeluis gave first theory of chemical catalysis, pointed out that an extract of malt known as DIASTASE catalyze hydrolysis of starch more efficiently than does sulfuric acid. Others like Justus leaving argued that biological processes are caused by action of chemical substances that were then called as “Ferments”. www.indiandentalacademy.com
  9. 9. ENZYMES EVERYDAY LIFE We can discover enzymes in our home! 1. Baking : Dough handling becomes easier making it less sticky this because of enzymes. 2. Noodles & Pasta : Enzyme called NOOPAZYME prevents pasta and noodles from being overcooked and resulting in a soft and sticky texture. 3. Beer: In Brewing enzymes speed up the process of fermentation. www.indiandentalacademy.com
  10. 10. 4. Novoshape : these enzymes used for processing fruits and vegetables. 5. Tooth paste with enzymes strengthens our mouth defence against bacteria by dissolving harmful microorganisms 6. Lipex : A lipase is used for removing fatty strains from lipstick and oil. www.indiandentalacademy.com
  11. 11. FOOD ENZYME CONCEPT • Given by Edward Howell • By eating raw food, work of enzymes is less • By reducing amount of food, we can contribute to higher enzyme potential. • He believes that mankinds change in diet from mostly uncooked to cooked foods has probably resulted in changes in structure of our GIT. www.indiandentalacademy.com
  12. 12. MODE OF ACTION • Enzymes work by lowering activation energy which the energy required by a system to initiate a particular process. • It is minimum energy required for a specific chemical reaction to occur. • As molecules approach their electron clouds repel each other. • To overcome this repulsion activation energy is required which provided by heat of system Translational Energy Vibrational Energy www.indiandentalacademy.com Rotational Energy
  13. 13. Enough energy is available Repulsion is overcome Molecules get close Attraction Rearrangement of bonds www.indiandentalacademy.com
  14. 14. Activation Energy & Enzymes www.indiandentalacademy.com
  15. 15. ENZYMES IN RELATION TO ΔG & LAW OF CONSERVATION OF ENERGY • All reactions catalyzed by enzymes must be spontaneous containing a net negative Gibbs free • energy. Given a particular sat of conditions, and products of a particular reaction (Including Net Energy) must be identical independent of specific individual pathway taken from beginning point to end point. This is by law of conservation of energy. www.indiandentalacademy.com required
  16. 16. COENZYMES Protein Part + Non Protein Part Apoenzyme Coenzyme HOLOENZYME www.indiandentalacademy.com
  17. 17. EXAMPLES Coenzyme Group Transferred 1. Biotin CO2 2. Coenzyme A Acyl Group 3. Tetrahydrofolate ‘C’ Group METALLOENZYMES Enzymes which require certain metal ions for their activity. EXAMPLES 1. Zn 2. Mg 3. Cu 4. Fe Carbonic an Hydrase Hexokinase Tyrosinase Cytochrome oxidase www.indiandentalacademy.com
  18. 18. ACTIVE SITE OF ENZYME Area of enzyme where catalysis occurs. Salient features : • It occupies only a small portion of enzyme • Situated in a cleft of enzyme molecule • Substrate binds to enzyme at active site by noncovalent which are hydrophobic in nature www.indiandentalacademy.com
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  20. 20. MAJOR CLASSES 6 Classes (by IUBMB) • Oxidoreductases ALC E.g. Alcohol + NAD Dehydrogenase Aldehyde + NADH2 • Transferrases : Transfer of a group other than hydrogen between a pair of substrate E.g. α Ketoglutarate Transferase • Hydrolases : Hydrolases of ether, ester, peptide, etc. E.g. Amylase, Pepsin www.indiandentalacademy.com
  21. 21. • Lyases : Removal of groups from substrates by mechanism other than hydrolysis, leaving double bonds. These enzymes act on C-C, C-O, C-N, C-S etc., E.g. L – Malate hydrolase • Isomerases : Conversion of optical or geometric isomers E.g. L – alanine isomerase • Ligases : Linking together of to compounds www.indiandentalacademy.com
  22. 22. THEORIES Lock and Key hypothesis. It states that three dimensional structure of active site of enzyme is complementary to the substrate. Thus enzyme and substrate fit each other similar to lock and key. key will fit only to its own lock. www.indiandentalacademy.com That
  23. 23. KOSHLAND’S INDUCED FIT THEORY • Substrate fixes at s shallow groove of enzyme but at present, alignment is not correct. • Fixing of substrate induces structural changes in enzyme, now substrate correctly fits into active site of enzyme. • Substrate analogue can’t bind properly. www.indiandentalacademy.com
  24. 24. Enzyme Kinetic : • Velocity or rate of enzyme reaction is assessed by rate of change of substrate to product per unit time. The rate of reaction is directly proportional to concentration of reacting molecules where K1 A+B Keq K2 = C+D K1/K2 = [C] [D] / [A] [B] www.indiandentalacademy.com
  25. 25. FACTORS INFLUENCING ENZYME ACTIVITY Effect of enzyme concentration Rate of reaction α enzyme sufficient substrate is present. Important: Concentration of enzymes does not effect keq but increases rate of reaction www.indiandentalacademy.com concentration when
  26. 26. Effect of Substrate Concentration Velocity increases on increasing substrate concentration in initial phases but curve flattens afterwards because at higher concentration all enzyme molecules are saturated. Michaeli’s Constant : (Km) Independent of enzyme concentration and denotes affinity of enzyme for substrate www.indiandentalacademy.com
  27. 27. Effect of concentration of products On increasing product concentration rate of reaction is decreased. Effect of pH Each enzyme have optimal pH, on both sides of which velocity decreases. between 6-8. Usually enzymes have optimal pH Exceptions are pepsin (1-2), phosphatase (9-10) and Acid phosphatase (4-5). www.indiandentalacademy.com Alkaline
  28. 28. Effect of Temperature Firstly on increasing temperature, rate increases upto maximum and this temperature is called as optimal temperature. REASON : As temperature increases more molecules get activation energy or more molecules are at increased rate of motion, so their collision probabilities are increased and therefore velocity increases. But, when temperature increases above 50°C, because of loss of tertiary structure of proteins, velocity decreases. Most human enzymes have optimal temperature of 37°C. www.indiandentalacademy.com
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  30. 30. ENZYME INHIBITION A. Competitive Inhibition Here inhibitor molecules are competing with the normal substrate molecules for attaching with the active site of the enzyme. In this inhibitor will be a structural analogue of the substrate. Clinical Significance Pharmacological action of many explained on basis of competitive inhibition. www.indiandentalacademy.com drugs may be
  31. 31. Examples Sulfonamides, methotrexate, Dicoumarol. These are antibacterial agents. Bacteria synthesize folic acid by combining PABA with glutamic acid. Bacterial wall is impermeable to folic acid. Sulpha drugs being structural analogues of PABA will inhibit folic acid synthesis in bacteria and they die. www.indiandentalacademy.com
  32. 32. B. Non competitive Inhibition Non competitive inhibitors don’t have structural resemblance to substrate. They bind to site of enzyme other than the active site. When substrate binds with it, no product is formed. Clinical Significance • Cyanide inhibits, cyto-oxidase • Fluoride inhibits Enolase www.indiandentalacademy.com
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  34. 34. Comparison between competitive & Non competitive Inhibition Acting on Competitive inhibition Active site Non competitive Inhibition May or May not Structure of Inhibitor Inhibition is Substrate Analogue Reversible Km Increases Unrelated Molecule Generally irreversible No Change Vmax No change Decreases www.indiandentalacademy.com
  35. 35. ALLOSTERIC INHIBITION • Here inhibitor is not a substrate analogue • Inhibitor binds to allosteric site causing structural change in active site. Therefore substrate doesnot fits into that site and therefore no product is formed. Clinical Significance Allosteric enzymes used in our body for regulating metabolic pathways such enzymes called as key enzymes. www.indiandentalacademy.com
  36. 36. Examples HMGCOA – reductase : Cholesterol synthesis Phosphofructokinase : Glycolysis www.indiandentalacademy.com
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  38. 38. SUBSTRATE SPECIFICITY • The non covalent forces through which substrates and other molecules bind to enzymes involve Vanderwall forces, electrostatic, H-bonding and hydrophobic interactions. • In general, a substrate binding site consists of cleft or indentation on surface of enzyme molecules i.e. complementary in shape to substrate. • As enzymes have their inherent chirality (Proteins consists of only L amino acids) form asymmetric active sites therefore enzymes are specific in binding chiral substrates. This is called as “STEREOSPECIFICITY” www.indiandentalacademy.com
  39. 39. REGULATION OF ENZYME ACTIVITY Two Ways : 1. Control of enzyme availability Amount of an enzyme in a cell depends on both rate of synthesis and its rate of degradation. Examples E.coli grown in absence of lactose lack enzymes to metabolize this sugar but on exposure to lactose, within a few minutes, bacteria start synthesizing enzymes required to utilize lactose. www.indiandentalacademy.com
  40. 40. 2. Control of enzyme activity This is regulated by conformational or structural alterations. Rate of enzyme catalysed reaction α concentration of E-S complex which in turn varies with substrate affinity. concentration and enzyme substrate binding Therefore catalyst activity of enzyme controls the variation of its substrate binding affinity. Examples Hb oxygen binding capacity is regulated by binding of ligands like O2, CO2 and H+. www.indiandentalacademy.com
  41. 41. MIXED INHIBITION OR NONCOMPETITIVE INHIBITION • Not to confuse it with uncompetitive inhibition • If both enzyme and E-S complex bind inhibitor, following model results. E+S K1 K2 ES + P+E + I K3 I K4 EI K5 ESI No Reaction www.indiandentalacademy.com
  42. 42. Both inhibitor binding steps are assumed to be at equilibrium but with different dissociation constants : [E] [I] K4 = -----------[EI] & K5 = [ES] [I] -----------[ESI] www.indiandentalacademy.com
  43. 43. BISUBSTRATE REACTIONS We have been concerned with reactions involving only one substrate. Yet enzymes reaction involving 2 substrates and yielding 2 products are there. A+B E P+Q www.indiandentalacademy.com
  44. 44. These type of reactions account for 60% of known bichemical reactions. Almost all of these so called bisubstrate reaction are either transferrase reactions in which enzyme catalyses the transfer of a specific functional group ‘X’ from one of substrate to other : P–X+B E P+ B–X Or oxidation reduction reactions. www.indiandentalacademy.com
  45. 45. Types of Bisubstrate Reactions : • Sequential reactions All substrates must combined with enzyme before a reaction can occur and products be released are called as sequential reactions. • In these group being transferred, X is directly passed from A to B yielding P & Q therefore these reactions also called as single displacement reactions. A B K2 K1 E P EA K3 EAB Q K5 K4 EPQ www.indiandentalacademy.com EQ
  46. 46. Ping Pong Reactions One are more products are released before all substrates have been added. In this First Stage Functional group ‘X’ of first substrate ‘A’ is displaced from substrate by enzyme ‘E’ to yield first product ‘P’ and ‘A’ stable enzyme form ‘F’ in which ‘X’ is tightly bound to enzyme (Ping). www.indiandentalacademy.com
  47. 47. Second Stage ‘X’ displaced from enzymes by second substrate ‘B’ to yield second product Q & therefore generating original form of enzyme (Pong). Such reaction also called as double displacement reactions. Examples Chymotrypsin, Transaminases act by ping pong reactions. www.indiandentalacademy.com
  48. 48. Catalytic Mechanisms Catalyses is a process that increases rate at which a reaction approaches equilibrium. Types A. Acid base catalysis : Acid catalysis : process in which partial proton transfer from a Bronsted acid (a species that can donate protons) lowers free energy of a reactions transition state. Base catalysis : Partial H+ abstraction by a Bronsted www.indiandentalacademy.com Base (Species that can combine with H+).
  49. 49. Examples • Mutarotation of glucose (Glucose molecule can assume either of 2 anomeric forms α or ß – D – through intermediacy of its linear form). • Hydrolases of peptides and esters www.indiandentalacademy.com glucose
  50. 50. B. Covalent Catalysis Involves rate acceleration through the transient formation of a catalyst substrate covalent bond. E.g. Decarboxylation of Acetoacetate. C. Metal Ion Catalysis Nearly 1/3rd of all known enzymes require the presence of metal ions for catalytic activity. 1. Metalloenzymes : Contain tightly bound metal ions like Fe2+, Fe3+, Cu2+, Zn2+ 2. Metal activated enzymes : loosely bind metal ions + from solutions. E.g. Nawww.indiandentalacademy.com , K+, Ca++.
  51. 51. LYSOZYME : MODEL OF ENZYME ACTION • A number of lysozymes found in nature in human tears and egg white. • It is antibacterial because it degrades the polysaccharide that is found in cell walls of many bacterial. • Globular proteins with a deep cleft across part of its surface, in which substrate fixes. • H bands form with > C=O groups of several peptide bonds. www.indiandentalacademy.com
  52. 52. • Hydrophobic interactions may help hold the substrate in position. • When lysozyme and substrate unite, both are slightly deformed. Fourth hexose in the chain (ring # 4) becomes twisted. This imposes strain on C-O bond and at this point only polysaccharide gets broken. • A molecule of water is inserted between these two hexoses which brakes the chin. www.indiandentalacademy.com
  53. 53. • As atoms have been distorted from their normal position therefore energy needed to break bond between them is lowered down. • Binding of substrate induces a small movement (0.75 A°) of certain aminoacid residues so the cleft closes slightly over its substrate. So the lock as well changes shape as two are brought together. www.indiandentalacademy.com as key
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  56. 56. ACTION OF RESTRICTION ENDONUCLEUS Cuts one strand of DNA double helix at one point and second strand at a different. Separated pieces have single stranded sticky ends which allow complementary pieces to combine. New joined pieces are stabilized by DNA ligases. www.indiandentalacademy.com
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  58. 58. Clinical Importance / Pathological Importance of Enzymes : • Presence of numerous enzymes in serum indicates that cellular or tissue damage has occurred • SGOT & SGPT indicates liver disease. • LDH and CK indicates myocardial infarction • Following myocardial infarction LDH level rises by 2448 hours reaching a peak by 2-3 days and return to normal in 5-10 days. www.indiandentalacademy.com
  59. 59. • CPK found in heart, skeletal muscle and brain therefore if its level rises with in 6 hours of injury to these tissues. • Pancreatin enzymes used in treatment of skin cancer (Recent) • A proteolytic enzyme called “Miracle Enzyme” also called as serrapeptase, used for opening clogged up arteries (Recent). www.indiandentalacademy.com
  60. 60. Genetic diseases graphics to accompany chap 4 of BRS path 2nd edition note: some diseases omitted because there were no graphics available or the diseases were obvious (Cystic fibrosis) Suhas Radhakrishna www.indiandentalacademy.com 5-11-03
  61. 61. Lysosomal 1: Tay Sachs CNS degeneration Mental/motor deterioration Cherry red spot on macula Blindness Death before 4 years old usually Enzyme Deficiency: Hexosaminidase A Accumulation: Gm2 ganglioside www.indiandentalacademy.com
  62. 62. Type II (infantile) CNS involvement, Lysosomal 2: Gaucher disease Death before 1 year old Type III (juvenile) also has CNS complications Wrinkled tissue paper cytoplasm Gauchers in spleen, LN, liver, bone marrow (RE system) Femoral head /long bone erosion Mild anemia Enzyme Deficiency: Glucocerebrosidase / B-D glucosidase Accumulation: Glucocerebroside / Glucosylceramide Note: the first is from BRS the second from Qbank, both seemed to be used by emedicine www.indiandentalacademy.com
  63. 63. Lysosomal 3: Niemann-Pick Disease Fever, neuro deterioration 50% of pts : cherry red spot Foamy histiocytes in liver, spleen, LN, skin Hepatosplenomegaly Enzyme deficiency: Sphingomyelinase Accumulation: Sphingomyelin Anemia www.indiandentalacademy.com Death by age 3
  64. 64. Lysosomal 4: Hurler syndrome Progressive mental retardation Corneal clouding “Gargoyle-like” facies (terrible name!) Dwarfism Stubby fingers Death by age 10 Enzyme deficiency: alpha-L iduronidase www.indiandentalacademy.com Accumulation: Heparan sulfate, dermatan sulfate
  65. 65. Glycogen Storage 1: von Gierke disease Accumulation of glycogen in liver and kidney => hepatomegaly Hypoglycemia Enzyme deficiency: Glucose 6 phosphatase Accumulation: Glycogen www.indiandentalacademy.com
  66. 66. Glycogen storage 2: Pompe disease Muscle hypotonia Splenomegaly cardiomegaly Death before age 3 of cardiorespiratory failure Enzyme deficiency: alpha-1,4 glucosidase Accumulation: Glycogen Intractable Hypoglycemia www.indiandentalacademy.com
  67. 67. Glycogen Storage 3: Cori Disease Glycogen accum in heart Glycogen in liver -> Hepatomegaly hypoglycemia Glycogen in skeletal muscle Stunted growth Enzyme deficiency: Amylo-1,6-glucosidase Accumulation: Glycogen www.indiandentalacademy.com
  68. 68. Glycogen Storage 4: McArdle Syndrome Enzyme deficiency: Muscle phosphorylase Accumulation: Glycogen Muscle cramps and weakness after exercise www.indiandentalacademy.com
  69. 69. Carbohydrate 1: Classic galactosemia Enzyme deficiency: Galactose-1-phopshate uridyl transferase Accumulation: Galactose -1-P Mental retardation Cirrhosis www.indiandentalacademy.com Failure to thrive
  70. 70. Decreased pigmentation of hair, eyes, skin Amino Acid 1: PKU Progressive mental deterioration Seizures Hyperactivity Neuro problems Musty/mousy body odor Enzyme deficiency: Phenylalanine hydroxylase Accumulation: Phenylalanine and its degradation products phenylpyruvic acid and phenylacetic acid www.indiandentalacademy.com
  71. 71. Amino Acid 2: Alkaptonuria Enzyme deficiency: Homogentisic oxidase Accumulation: Homogentisic acid Ochronosis – dark pigmentation of fibrous tissues and cartilage Can affect the joints and the heart Dark/Black urine www.indiandentalacademy.com
  72. 72. Amino Acid 3: Maple Syrup Urine Disease Urine smells like maple syrup Can lead to neonatal death if untreated! Enyzme deficiency: branched chain alpha-keto acid dehydrogenase constituent proteins www.indiandentalacademy.com
  73. 73. X linked 1: Hunter Syndrome Mild mental retardation Retinal degeneration Micrognathia Joint stiffness Hepatosplenomegaly Cardiac lesions Enzyme deficiency: L-Iduronosulfate sulfatase Accumulation: Heparan sulfate, dermatan sulfate www.indiandentalacademy.com
  74. 74. PERIODONTAL DISEASE Cathepsin C enzyme which destroys diseased cells and eliminate infection in mouth. ORAL ENZYMES IN TREATMENT FOR HEPATITIS C Four Methods were checked for the treatment of hepatitis C • Enzyme Combination : Phlogenzym • Interferon • Ribovarin • Liver supplements www.indiandentalacademy.com but results with phlogenzyms were better
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  76. 76. ENZYME THERAPY VS CANCER Contains proteolytic enzymes which break down • Role of enzyme therapy for cancer : 1. Restore body internal environment : Keeps body pH to be slightly alkaline, eliminate body wastes and facilitate excretion. 2. Enzyme breakdown tumour cells 3. Enzymes purify blood by eliminating toxins. 4. Enzymes can reverse tumour from malignant to benign and can induce Apoptosis in tumour cells. www.indiandentalacademy.com
  77. 77. ENZYME DETECTION Techniques 1. ELISA – for alkaline phosphatase, Horseradish peroxidase. 2. Phosphatases - Alkaline Phosphatase 1. 2. Azo Dye method 3. - Gomori Calcium method Using simple naphthols Acid phosphatases 1. GOmori Pb method 2. Azo www.indiandentalacademy.com dye method
  78. 78. 3. Esterases - Non specific : Carboxy, Aryl, Acetylesterases : 1. 2. - α naphthyl acetate method Indoxy acetate method Specific : Acetylcholinesterases, lipases 1. Tweens method 2. Filipe & Lake method www.indiandentalacademy.com
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