Enzyme biochemistry


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Enzyme biochemistry

  1. 1. Enzymes are protein catalysts that ↑se the rate of reaction 33 without being changed in the overall process. Thus enzymes direct all metabolic events.
  2. 2. a. b. Enzymes are neither consumed nor produced during the course of a reaction. Enzymes only expedite the reaction & do not cause reactions to take place.
  3. 3. a. b. c. Enzymes are invariably proteins Highly specific Enzymes function within a moderate Ph & Temp range E + S → ES → E + P
  4. 4. The names of enzymes in many cases end in “ ase” which is preceded by the name of its subtrate e.g. sucrase, lipase, etc. in other cases their name describes the action of an enzyme e.g. transmethylase. In other case their names do not all point out their substrate or action e.g. pepsin, trypsin. International union of biochemistry & molecular biology drafted specific rules for the classification & nomenclature of enzymes.
  5. 5. In this system each enzyme has been assigned as 4-digit classification number along with a systemic name which indicates the catalysed reaction. The digits represent the “class” “sub class” & “sub-sub class” An i.u of an enzyme is defined as the qty. of enzyme needed to transform 1.0 micromole of its substrate to the product/min at 30 ⁰ & optium pH.
  6. 6. Measure of enzyme activity are the specific activity and the Katal. The specific activity is the no. of units of enzyme activity/mg of enzyme protein. The Katal is the amount of enzyme activity that transform one mole of its substrate to the product/second.
  7. 7. There are six main classes of enzymes, each one of these is further sub ÷ed x subclasses & sub-subclasses. The main classes are the following: I. Oxidoreductases II. Transferases III. Hydrolases IV. Lyases V. Isomerases VI. ligases 
  8. 8. These enzymes catalytyse oxidationreduction reactions by transfer of electrons. This group is further sub ÷ed x 6 subgroups i.e.
  9. 9.  In reactions catalyzed by these enzymes, oxygen is added to H-atoms removed from the substrate for e.g. ascorbic acid oxidase.
  10. 10. O= C I HO – C II HO – C O I H - C I HO – C – H I CH2OH Ascorbic acid ( vit C) ½ O 2 H2 o O= C I O– C O I O– C I H - C I HO – C – H I CH2OH Dehydroascorbic acid
  11. 11. These enzymes catalyze the removal of H 2 from a substrate & can use either oxygen or substances like methylene blue as H2 acceptors. e.g. Glucose oxidase which catalyses the oxidation of glucose to gluconolactone
  12. 12. H– C - OH I H – C - OH I HO – C - H I H - C - OH I H – C I CH2OH Glucose O2 O C=O I H C - OH H2 o – I HO – C – H I H - C - OH I HO – C I C–H I CH2OH O Gloconolactone
  13. 13. These enzymes catalyse the removal of hydrogen from the substrate & are unable to use oxygen as hydrogen acceptor the Hacceptors take the H-atoms e.g. NAD+ , NADP+ and FAD all these three substances act as co-enzymes for their respective enzymes.
  14. 14. COOH COOH I I Lactate dehydrogenase HO - C - H + NAD+ C = O + NADH + H+ I I CH3 CH3 Pyrimic acid Lactic acid
  15. 15. There are 2 enzymes in this class called peroxidase + catalase. Catalyze the decomposition of H2 O2. Catalase. Catalyze the following reaction: 2H2O2 2H2O + O2
  16. 16.  These enzymes catalyze the incorporation of molecular O2 x the substrate. An e.g. is the conversion of phenylalanine to tyrosine by the enzyme phenylalanine hydroxylase.
  17. 17. H H I I CH2 - C – COOH C – COOH O2 H2 o CH2 I I I I + NADPH + H+ -----------→ NH2 NH2 + NADP+ I OH Phenylalamine Tyrosine
  18. 18. These enzymes catalyze the reduction of their substrate by adding H-atoms. e.g. glutathione reductase which catalyze the conversion of oxidised glutathione to reduced glutathione.
  19. 19. - Glu - Cys - Gly I S I +NADPH+H+ S I - Glu - Cys - Gly 2 - Glu - Cys – Gly + NADP+ I SH
  20. 20. 1. these being about an exchange of functional groups such as phosphates, amino, acyl & methyl b/w 2 compounds diff. types of transferases are: Transaminases: These catalyze the exchange Of –NH2 gp b/w an amino & a ketoacid. The ketoacid becomes amino acid & the amino acid becomes ketoacid.
  21. 21. COOH І H2NCH + І CH2 І CH2 І COOH Glutamic acid COOH І C=O І CH2 І COOH Glutamic – Oxaloocetic Oxaloocetic acid Transaninase (Got) COOH І C=O + І CH2 І CH2 І COOH Ketoglutamic acid COOH І H2N C H І CH2 І COOH Aspartic acid
  22. 22. COOH І H2NCH + І CH2 І CH2 І COOH Glutamic acid COOH І C=O І CH3 Pyruic acid Glutamic Pyruic Transaninase (GPT) COOH І C=O + І CH2 І CH2 І COOH Ketoglutamic acid COOH І H2 N C H І CH3 І Alanine acid
  23. 23. 2. Phosphotransferases(kinases):These catalyse the transfer of PO4 gps. & are called kinases. e.g. hexokinase which catalyze the reaction, Glucose + ATP → glucose 6 PO4+ ADP
  24. 24. 3. Transmethylases: These catalyze the transfer of methyl gps. e.g. conversion of noradrenaline to adrenaline
  25. 25. CHOH – CH2 – I OH - N -H I H CH3 CHOH – CH2 – I OH - I OH Noradrenaline I OH Adrenaline N -H I CH3
  26. 26. 4. Transpeptidases: These catalyze the transfer of α. α or peptides e.g. formation of hippuric acid from benzyl CoA + glycine
  27. 27. O = C – S – CoA I + H2N – CH2 – COOH glycine Benzyl CoA O = C – N - COOH I I H + CoA - SH Benzyl glycine or Hippuri acid
  28. 28. Transacylases: These catalyze the transfer of acyl gps. e.g Choline acetyltransferase which catalyzes the synthesis of acetylcholine Acetyl – CoA + Choline → acetylcholine + CoA 5.
  29. 29. Catalyze hydrolosis i.e add H2o molecule to the substrate + Simultaneously decompose it. Various sub-groups are: 1. Protein hydrolyzing enzymes i.e proteinases or proteases or proteolytic enzymes A. Expopeptidases: Catalyze the hydrolysis of terminal peptide bonds B. Endopepridases: Attack the centrally located peptide bonds 
  30. 30. A. i. Exopeptidases: further sub÷ed x:Polypeptidases : 2 types Aminopolypeptidases: Occurs in the intestinal juice & attacks the protein molecule from the side containing a free amino gp. Yielding an amino acid + peptide smaller in size by one α .α reside b. Carboxypolypeptidases: acts in the same way as amino polypeptidase + attacks from the side containing a free carboxyl gp + is present in pancreatic juice. a.
  31. 31. ii. iii. Triptidases Act on tripeptide liberating 3 α .α Dipeptidases act on dipeptides liberating 2 α .α
  32. 32. B. Endopeptidases e.g. pepsin, trypsin, chymotrypin + elastase. All these effect hydrolysis at particular α .α residues
  33. 33. 2. Carbonhydrases catalyzxe the hydrolysis of the glucosidic bonds e.g. enzyme amylase converts starch to maltose. Maltose is further hydrolysed by the enzyme maltase to glucose. Sucrase, lactase & cellulase also are e.g. of this gp. of enzyme.
  34. 34. 3. i. ii. iii. Lipid hydrolyzing enzymes: e.g. Lipases: Act on triglycerides or neutral fats to liberate glycerol, F.A + monoglycerides + diglycerides. Pancreatic lipase is very impt. In the digestion of fats. Cholesteryl esterase: Hydrolyses cholesterol esters. Phospholipase: Also known as lecithinases + splits lecithins as well as cephalins
  35. 35. 4. Deaminases or aminohydrolases: Include adenases + guanase which catalyze the following reactions:Adernine +H2O → Hypoxanthine + NH3 Guanine +H2O → xanthine + NH3
  36. 36. 5. Deamidases or amidohydrolases: Catalyze the hydrolysis of amides + include urease, arginase, glutaminase + asparginase which catalyze the following reactions respectively:Urea + H2O → Co2 + 2NH3 Arginine + H2O → orinithine + urea Glutamine + H2O → Glutamine acid + NH3 Aspargine + H2O → Aspartic acid + NH3
  37. 37. Other ester hydrolyzing enzyme:grouped x 2 types A. Phosphatases B. Miscellaneous 6.
  38. 38. A. i. ii. Phosphatases Phosphomonoesterases: occur in blood, plasma, bone, prostate, kidney, RBC, milk + intestinal mucosa, e.g. the hepatic enzyme glucose 6-PO4 – PO4 ase are which catalyze the reaction glucose 6PO4 + H2O → glu. + Phosphoric acid Phosphodiesterase: it splits off one PO4 gp of diesters.
  39. 39. iii. Phosphorylase: Adds inorganic PO4 to split a bond e.g. glycogen Phosphorylase present in hepatocytes + SK. Muscle fibres which catalyze the reaction. Glycogen + H3PO4 → Glucose 1-PO4 iv. Pyrophosphatase:- this enzyme hydrolyze pyrophosphates to orthophosphate PPi + H2O → 2 pi
  40. 40. v. vi. vii. Nucleases or polynucleatidases: Present in the intestinal juice + tissues these decompose nucleic acid i.e. DNA + RNA Nucleotidases: Occur in intestinal juice + tissues + hydrolyses mononucleotides to nucleosides + H3PO4 Nucleosidases: Catalyze the reaction Nucleoside + H3 PO4→ free nitrogenous base + pentose PO 4
  41. 41. B. i. Miscellaneous ester hydrolyzing enzymes:Cholinesterase: 2 types i. ii. ii. True type : Hydrolyzes only acetylcholine to ecatic acid + choline. Pseudo Type : variety is not specific + hydrolyzes other related substrates as well. Sulfatase: hydrolyses sulfate esters e.g. phenol sulfatase which is present in kidney + brain catalyzes the hydrolysis of phenol sulfate to phenol + H2 SO4
  42. 42. iv. Lyases: Catalyze the addition of NH3, H2O or CO2 to double bonds or the removal of these from double bonds e.g. conversion of fumaric acid to Lmalic acid
  43. 43. COOH I funerase CH + H2O II HC I COOH Fumaric acid COOH I HOCH I CH2 I COOH L- Malic acid Carbonic an hyderase present ion red blood cell gastric mucosa renal tubules catalyzes the reaction H2 O + Co2 H2 CO3
  44. 44. v. Isomerases: Catalyze the transfer of gps. With in molecules to yield isomeric forms of the substrate . eg. Glucose 6 - PO4 fructose 6- PO4 phosphohexose isomerase other e.g are reductases, oxidases
  45. 45. vi. Ligases: Catalys reactions joining 2 mol by forming C- O, C – S, C - N & C - C bond
  46. 46. e.g. CH3 I C = O + CO2 + ATP I SCOA Acetyl CoA Acetyl – CoA carboxylase COOH I CH2 + ADP +P1 I C=O I S - CoA Malonyl coa
  47. 47. 1. Protein nature: Enzymes are protein catalysts ↑es the velocity of a chemical reaction + are not consumed during the chemical reaction their catalytic activity depends upon the integrity of their structure as proteins e.g. when boiled with acid or incubated with trypsin that will cleave the polypeptide chain & their catalytic activity is lost.
  48. 48. Showing primary backbone structure is reqd. Disruption of characteristics folding of polypeptide chain of a native enzyme protein by heat & by exposure to extreme pH or temp. with other denaturing agents the catalytic activity is lost . Enzymes like other protein have mol. wt. ranging from 12,000 to over a million.
  49. 49. 2. Chemical nature: Some enzymes consist only of polypeptides & contain no chemical gp. Other than α. α resides e.g. “ pancreatic ribonuclease”. Other enzymes require an additional chemical component for their activity called a “ co factor” which may be inorganic like Fe+2, Mn +2 or Zn 2+ ions.
  50. 50.   Organic:- Called co enzymes. Some enzymes require both coenzymes & one or more metal ions which may be loosely or tightly bond to the protein permanently. Prosthetic group:- The co-enzymes or metal ions when tightly & permanently bond that do not dissociate from the enzyme is known as prosthetic group e.g. biotin.
  51. 51. 3. Holo Enzymes:- Refer to the active enzyme with its non protein component whereas the enzyme without its nonprotein moiety is termed as “ apoenzyme” & is inactive if the non protein moiety is metal ion such as Zn++ is called a “ co factor” if it is a small organic mol. It is termed a “co enzyme”.
  52. 52. 4. Active sites:- enzyme mol. Contain a special pocket or clefty called the active site. Which contains α. α side chains that participate in substrate binding & catalysis. The substrate binds the enzyme forming an ES complex. ES is converted to an enzyme product (EP) that subsequently dissociates to enzyme & product. E + S → ES → EP → E+P
  53. 53. 5. Catalytic efficiency : Enzyme catalysed reaction are highly efficient. The number of molecules of substrate converted to product per enzyme molecule per second is called the turn over number or K cat.
  54. 54. 6. 7. Specificity: Enzymes are highly specific interacting with one or a few substrates & catalysing only one type of chemical reaction. Regulation: Enzyme activity can be regulated i.e. ↑ed or ↓ed.
  55. 55. 8. Location within the cell:- Many enzymes are localized in specific organelles within the cell which serves to isolate the reaction substrate or product from other competing reaction , providing favorable environment for the reaction & organizes the no. of enzymes present in the cell x useful pathways.
  56. 56. A. Energy changes occurring during a reaction All chemical reaction have an energy barrier separating the reactants & the products which is called “the free energy of activation”. This barrier is the energy difference between that of the reactants & a high energy intermediate that occurs during the formation of product. A T* B
  57. 57. T* uncatalyzed T Catalysed Free energy A Reactants imitate state Product B Find state i. Free energy of reaction ii. Rate of reaction iii. Alternate reaction pathway Progress of reaction
  58. 58. i. Free energy of reaction: The peak of energy is the diff. in free energy between the reactant & T where high energy intermediate is formed during the conversion of reactant to product which of the high free energy of activation the rates of uncatalyzed chemical reactions are often slow.
  59. 59. ii. Rate of reaction : It is determined by the no. of energized molecules. In general the lower the free energy of activation, the more molecules have sufficient energy to pass through the transition state. & thus the faster the rate of the reaction.
  60. 60. iii. Alternate reaction pathway: An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower free energy of activation. Enzymes do not change the activation. Enzymes do not change the equilibrium of the reaction but accelerate the reaction which with equilibrium is reached.
  61. 61. B. Specificity : Enzyme exclusively binds to & react with particular molecules or classes of molecules that are substrates for the reactions they catalyse . They specificity of enzyme action has been explained by two theories:a. Lock & key theory b. Induced fit theory
  62. 62. a. Lock & key theory:- The active site of enzyme is complementary in confirmation to the substrate, so that the enzyme + substrate recognize each other.
  63. 63. Substrate Enzyme Active site Enzyme substrate complex Product
  64. 64. b. Induced fit theory:- The enzyme changes shape upon binding the substrate, sop that the confirmation of substrate & enzyme protein are only complementary after the binding reaction. the important feature of this model is the flexibility of the region of active site.
  65. 65. According to this the active site doesn't process a rigid performed structure on enzyme to fit the substrate. On the other hand the substrate during its binding induces conformational changes in the active site to attain the final catalytic shape & form.
  66. 66. This explains several matters related to enzyme action such as : • enzyme become in active on denaturation • Saturation kinetics • competitive inhibition • All osteric modulation
  67. 67. Active site Enzyme Substrate Initial Binding of Enzyme with substrate Product fit b/w enzyme & substrate Enzyme Product
  68. 68. • • • Functional groups of enzyme Cofactor substrate
  69. 69.  1. The active site of the enzyme may furnish R groups of the Sp α.α resides that are: Good proto….. Or acceptors. Such general acid or base gps. are powerful catalysis for many organic reactions in aqueous system” proton donor” gps. may be – COOH, +NH3 – SH + “ proton acceptors” may be - COO- , - NH2 – S
  70. 70. 2. 3. Nucleoplinlic gps or enzyme may participate in reaction e.g proteolytic enzymes i.e trypsin chymotrypsin, elastase. Some enzymes bond convalatlly which substrate to form ES complex & form products more rapidly
  71. 71. Co enzymes
  72. 72.   Coenzymes are defined as heat stable, low molecular weight organic compounds required for the activity of enzymes. Most coenzymes are linked by now covalent forces. Those which form covalent bonds are prosthetics gps.
  73. 73.  1. 2. 3. 4. Enzyme that require co enzymes catalyze following reactions:Oxidoraduction Isomerization Group transfer reactions Reactions resulting in formation of covalent bonds.
  74. 74. Many enzymes are derived from ….. These compounds are recycled and are needed only in catalytic amount. Coenzyme function as substrate in twosubstrate reactions being bond momentarily to the enzyme during catalysis. They are chemically altered during the course of reaction & are recovered to their original forms by same or another enzyme.
  75. 75. Classificati on of coenzymes
  76. 76. a. Nicotinamide adenine dineralotide –(NAD+) b. Nicotinamide adenine Phosphate (NADP+) c. Flavin mononucleotide (FMN) d. Flavin adernine dinucleotide (FAD)
  77. 77. a.b. Drived form Niocin & require it for their synthesis, small amount of Niocin is …. From tryptophane – essentail α. α c.d. Drived from riboflavin
  78. 78. a. b. Lipoic acid – also involved in acl gp transfer. Biopterinm – a pteridine containing compound & participate in certain hydroxylase .eg. Phynylalamine hydroxylase.
  79. 79. c. Coenzymes Q- is a gp of closely related compounds differing only in length of side chain. They can be synthesized in humans from ……. Pyrophosphate – an intermediate in cholesterol biosynthesis.
  80. 80. a. Thiamine pyrophosphate (TPP) is used for oxidatine decarboxylation of Ketoacids & in the trans ketolase catalyzed steps of the pentose phosphate pathway.
  81. 81. b. c. Pyridoxal phosphate:- involved in variety of reactions on amino acids e.g. racemization, decarboxylation, transamination, elimination of H2o or hydrogen sulfide . It is derived from vit. B6 Tera hydrofolic acid (FH4): is a carrier of one carbon fragments. It is derived from folic acid.
  82. 82. d. e. Coenzyme A(COA, COASH): takes part in acetyl + other acyl gp. Transfer + require vit. Pantothenic acid for its synthesis. Biotin is vit. Tightly bond to apoenz…. In an amide likage δ – amino gp. Of lysyl resid.. & involved in carboxylation reactions.
  83. 83. f. Cobamide coenzyme: contains cobalt bond in a porphyrin like ring system. It is involved in methyl transfer reactions. It is derived from cyanocarbalamin ( vit. B 12)
  84. 84. a. b. Adenosine triphosphate (ATP) can be a donor of phosphate, adenosine + adenosine monophosphate (AMP) for various purpose. Cytidine dipphosphate (CDP) is a carrior of phosphoeyl choline, dioxyl glycerols & other molecules during synthesis of phospholipids.
  85. 85. c. d. Uridine diphosphate (UDP) ia a carrior of monoisacehorides + their derivation in various reactions Phospho adenosine phospho sulfate is a sulfate donor in the synthesis of sulfurs containing monocopolysacharides & in detoxification of sterol steroid & other compounds.
  86. 86. Isoenzy mes
  87. 87. Isienzymes are the physically distinct forms of the same enzyme but catalyze the same chemical reaction or reactions & differ from each other structurally, electrophoretically & immunologically.
  88. 88. Isoenzymes are different molecular forms of enzymes that may be isolated from the same or different tissues. Their physical proportion are different because of genetically determined differences in amino acid sequence.
  89. 89. Different organs contain certain proportion of different isoenzymes. The pattern of isoenzymes found in plasma may there for same as a mean of identifying the site of tissue damage e.g. Creatine kinase – CK lactate dehydroganase – LDH Alkaline phosphatase - ALP
  90. 90. Many isoenzymes contain different subunits in various combinations e.g LDH. Various types are (H4) (H3M) (H2M2) (HM3) (M4) LDN –I, –5 LDH – 2, LDH – 3, LDH – 4, LDH
  91. 91. By electrophoresis Chemically Heat stability ii. Inhibition with urea iii. Reaction with changed substrate i.
  92. 92. In normal serum H3 M isoenzyme is present in highest conc. In an individual who has suffered a myocardial infarction, particularly H4 are elevated . The ↑se in H4 confirms the diagnosis that the patient suffered a myocardial infarction. (IHD)
  93. 93. Possible isoenzymes of CPK Type CK-1 CK-2 CK-3 Polypeptid e BB MB MM Electrophosat ic molarity Fast moving Intermediate Slow moving Tissue type Brain Hybrid Muscle type
  94. 94. Electrophoresis Ion exchange chronatography Radio immuno assay (RIA) Immuno inhibition
  95. 95. Types of Tissues CK isoenzyme s CK- BB Brain , bladder ↑ed level in condition CK - 1 Prostate, uterus, colon, stomach, lungs, thyroid Anoxia encephalopathy CNS – shock carcinomas. Placenta / utrine trauma. Co-poisoning acute & chronic renal failure. CK – MB Heart MI , angina CK – 2 SK. Muscle Ischemia, polymyositis CK – MM Sk. Muscle Mi CVA
  96. 96. Exists as a no. of isoenzymes Hepatic isoenzymes Intestinal isoenzymes Placental isoenzymes
  97. 97. Electrophoresis Chemical inhibition Heat inactivation
  98. 98. Major ALP isoenzyme in normal serum of healthy adult person is derived from liver. In growing children bone isoenzyme predominants. Hepatic isoenzymes ↑es in liver diseases. Bone isoenzyme ↑es due to osteoblastic activity & is normally elevated in children & adults over 50 years. chronic haemodialysis.
  99. 99. Placental isoenzyme ↑es in last 6 wks. Of pregnancy. Intestinal isoenzymes ↑es consumption of fatty meal. In disorders of GIT, cirrhosis of liver & in patients undergoing
  100. 100. Sources of plasma enzymes: 1. Plasma derived :- their activity is higher in plasma than cells. E.g. coagulation enzymes. 2. Cell derived:- Their activity is higher in cells overflow in plasma. a. Secretory – from digestive gland b. Metabolic – concerned with metabolism
  101. 101. Presence 1. 2. 3. 4. Cell derived enzymes Removal Normal turnover of tissue Intravascular inactivation( dilution, lack of substrate, coenzymes & protenase) Leakage through cell mem. Uptake by tissues with subsequent in activation Tissue necrosis Removal by RES. ↑se enzyme synthesis Excretion in urine of low mol. Unit. enzymes.
  102. 102. a. ↑sed release › Cell necrocesis › ↑sed cell mem. Permeability without cell necrosis › ↑sed enzyme production › An ↑se in cell no. producing enzymes
  103. 103. b. Impaired disposition/ secretion ↓ed formation of enzymes Genetic Acquired Enzyme inhibition (poising) Lake of cofactor.
  104. 104. 1. 2. 3. Diagnosis of different pathological conditions e.g. MI (CK,LDH, AGT) Prognosis: serial serum enzyme assay reqd. & change in serum enzyme level in ab…. Therapeutic use: streptokinase, an enzyme that facilitates the breakdown of clot, commonly used to dissolve a clot that causes MI.
  105. 105. Commonly carried enzyme assay in MI: Creatinie phosphokinase (CK) Aspartate transaminase (AST) Lactate dehydrogenase (LDH)
  106. 106. Catalyes the reaction Creatinine – P + ADP → creatine + ATP It is present in high [] in SK , muscle, myocardia+ brain. In small[] it is present in lung thyroid + kidneys. It is absent in liver. Normal value: 4- 60 iμ/L at 37 ⁰C
  107. 107. ↑es after 6hrs. Peak level – 24 – 30 hrs. Normal level – 2 – 4 days Serum CK level is a very sensitive indicator in early stages of myocardial ischemia
  108. 108. Enzym Start to e rise CK 4 – 8 hrs. Peak elevation 24 – 48 hrs. AST 6 - 8 hrs. 24 – 48 hrs. LDH 12 -24 hrs. 48 – 72 hrs. Duration 3–5 days 4–6 days 7 – 12 days
  109. 109.             Hepatocellular damage or ↑ed liver cell preamability Extrac hepatic or intrahepatic obstruction (being or malignant) Protein synthesis Alcohol abuse Trans aminases (SGOT) Oenithinie carboxyl transferase (OCT) Sobitol dehydrogenase Alkaline PO4ase 5-Nucleosidase Glutamyl transferase Pseudocholinestrase Glutamyl transferase
  110. 110. Acid PO4ase - prostate cancer Almie amino transferase – viral hepatitis, liver disease Alkaline PO4ase (ALP) – liver, bone diseases Amylase – acute pancreatitis Lipase - acute pancreatitis Isocitrate dehydrogenase – viral hepatitis
  111. 111. Serum LDH – unidespread malignancies Β – Glucouromidase in urine – cancer of urinary bladder
  112. 112. A metabolic pathway involves many enzymes functioning in a sequential manner. Control of the pathway is achieved through modulation of the activity of only one / few key enzymes i.e regulatory enzymes. A regulatory enzyme catalyze a rate – limiting chemical reaction that controls the overall pathway. It may also catalyze a chemical reaction unique to that pathway – committed step.  These enzymes which catalyze the rate limiting step or committed step of a pathway are under regulation. 
  113. 113. When the end product exceeds the steady – state level concentration, it inhibits the regulatory enzyme in an attempt to normalise the overall process.
  114. 114. Enzymes may be altered suppression. This regulation at the genetic level occurs. During various phases of reproduction, growth & development.
  115. 115. e.g. pyrnate dehydrogenase complex
  116. 116. e.g. Enzyme of glucogen breakdown
  117. 117.  Compartmentalization of enzyme system e.g. fatty acid synthesis occurs in the soluble fraction of cytoplasm. 6. Covalent modification 7. Non covalent/ allosteric modification 8. Induction & repression of enzyme synthesis
  118. 118. Some covalent chemical modifications are: i. Phosphorylation & de phosphosylation ii. Acetylation & de acetylation iii. Adenylytion & de adenylytion iv. Uridylylation & de uridylylation v. Methylation & de methylation 
  119. 119.  Phosphorylationis catalysed by proteinkinases & occurs at specific seryl residues& occasionally at through residues. These αα residues are not usually part of the catalytic site of the enzyme dephosphorylation is accomplished by phosphoprotein phosphophatases.
  120. 120.  The overall process of phosphorylation & dephosphorylation consists of an extracellular signal commonly referred to as first messenger e.g. hormones which combines with specific receptor on the cell membrane of target cell which produces an intracellular signal the 2nd messenger.
  121. 121. Depending on specific enzyme the phosphorylated form may be more or less active.
  122. 122. Enzymes are regulated by molecules called effectors modifiers or modulators, that binds non covalently at a site other than active site . They alter the affinity of enzyme for to substrate or modify the maximal catalytic activity of enzyme or both.
  123. 123.     Negative effecter: inhibit enzyme activity Positive effecter: increase enzyme activity H-omotropic effecter: substrate – severe as an effecter Hetrotropic effecter: effecter may be different from substrate.
  124. 124. Cells can regulate the amount of enzyme present by altering the rate of enzyme degradation or the rate of enzyme synthesis
  125. 125. Regulator event Typical effecter Results Time required Substrate inhibition Substrate Change in velocity Immediate Product inhibition Reaction product Change in Y `` or V max Aclosteric control Pathway end product Change in V `` max or KS Covalent modification Another enzyme Change in V `` max or Km Synthesis or degradation of enzyme Hormone Change in Hours to or the amount. days metabolite Of enzyme
  126. 126. Quantization of enzyme activity:- the rate at which the substrate changes to the product is directly proportional to : 1. Time 2. Enzyme concentration
  127. 127. 1. 2. 3. In zero order reaction, the rate , or velosity (V) is constant & is independent of the reactant concentration In first order reactions, the rate is proportioned to the reactant concentration In second order reactions, the rate is proportional to the product of the concentrations of the reactants.
  128. 128. Reaction model:  The enzyme sensibly combines with its substrate to form ES complex that subsequently breaks down to product, regenerating free enzyme. E +S ES → E+P K 1+ K1 + K2 = rate constant
  129. 129. Michaerlis describes how reaction velocity varies with substrate concentration Vi = V max [ ] Km + [s] Where Vi = initial reaction velocity Vmax = maximal velocity Km = Michaerlis constant = ( K-1+K2) K1 [s] = substrate concentration 
  130. 130.  1. 2. 3. Km is not equilibrium constant, it is ratio of constants Following assumptions are made in deriving equation Relatime concentrations of E + S Steady-state assumption Initial velocity
  131. 131. The conc. Of substrate is much greater than the concentration of enzyme [E] so that the amount of substrate bond by the enzyme at any one time is small.
  132. 132.   [ES] does not change with time – that is the rate of formation of ES is equal to that of the breakdown of ES ( E+ S + to E+P) In general, intermediate in a series of reaction is said to be in steady state when the rate of synthesis is equal to its rate of degradation.
  133. 133. Only initial reaction velocities are used in the analysis of enzyme reactions i.e the rate of the reaction is measured as soon as enzyme & substrates are mixed . At that time the concentration of product is very small and therefore the rate of back reaction from P to S can be ignored.
  134. 134. Characteristic of Km: The michaelis constant is characteristic of an enzyme & a particular substrate & reflect the affinity of the enzyme for that substrate. Km is numerically equal to substrate [ ] at with the reaction velocity is equal to ½ V max.
  135. 135. Km does not vary with the concentration of enzyme Small Km high affinity of enzyme for substrate Large Km low affinity of enzyme for substrate.
  136. 136. The rate of reaction is directly proportioned to the enzyme [ ] at all substrate [ ] e.g. if the enzyme [ ] is halved. The initial rate of reaction (V) is related to one half that of original. Km + Vmax may be influenced by pH, temp. & other factors.
  137. 137. In a metabolic pathway, Km values for enzymes that catyalyze the sequential reactions may indicate the rate – limiting step for the pathway the highest Km corresponds roughly to the slowest step.
  138. 138. When K >> K2 [ES] + [S] ES dissociating more after to yield E+S than to yield product When K2 >> K-1 The rate of dissociation of ES to E+S is small, so that products are usually formed.
  139. 139. When [S] >>Km The characteristic property of the turnover number for an enzyme can be invoked. This no. provides information regarding how many times it forms the ES complex & is regenerated by yielding P.
  140. 140. Order of reaction: At high [ ] of substrates the velocity of reaction is zero order. i.e constant & independent of substrate concentration. At low [ ] of substrate, the velocity of reaction is 1st order i.e proportional to substrate concentration.
  141. 141. Equation I = Vo Km Vmax I Vmax I V Km V max -I Km I V max O I [S] + [S] I
  142. 142. When the reaction velocity Vi is plotted against substrate concentration, [S}, it is not always possible to determine when V max has been achieved, because of the curve at high substrate [ ]. However of I/Vo plotted Vs I/[S ] a straight line is obtained. This plot is called line weaver burke plot & can be used to calculate Km & Vmax as well as determine mechanism of action of enzyme inhibitions.
  143. 143.   The activity is usually defined as that quantity of enzymes which catalyzes the conversion of one micromole of substrate to product per minute under defined set of optimal conditions. It is repressed in terms of μ/ml of biological specimen e.g. serum or μ/l.
  144. 144. 1. 2. 3. 4. Substrate concentration Enzyme 5. Temperature concentration 6. Effect of time Product 7. Effect concentration of activity 8. Effect of inhibitors ph
  145. 145.  All major factors that affect the rate of enzyme catalyzed reactions are of chemical interact. Good health requires not only that hundreds of enzyme catalyzed reactions to take place, but also that they proceed at appropriate rates. Failure to achieve this disturbs the homeostatic balance of our tissues with potentially profound consequences.
  146. 146. Maximal velocity: The rate or velocity of a reaction (V) is the number of substrate molecules converted to product per unit time & is usually suppressed as μ mole product formed per minute.
  147. 147. If the concentration of a substrate [s] is ↑ed while all other conclusions are kept constant the measured initial velocity Vi ↑ed to a maximum value Vmax. The velocity ↑ed as the substrate [] is ↑ed up to a point where the enzyme is said to be saturated with substrate.
  148. 148. Velocity V max V max 2 B A Km [S] C
  149. 149. At point A+B only a portion of enzyme present to combined with substrate. At point A or b ↑ing or ↓ing [S] with therefore ↑ or ↓ the amount of E associated with S as ES of Vi will depend on [s] at C all enzyme is combined with substrate so that further ↑se in [S], although it ↑es the frequency of collision b/w enzyme & substrate, cannot result in ↑ rate of reaction since no free enzyme is available to react.
  150. 150.  The initial rate of reaction is the rate measured before sufficient product has been formed to permit the severe reaction to occur. The initial rate of reaction catalyzed by enzyme is always proportional to the concentration of enzyme. ↑se in the concentration of enzyme ↑se the rate of reaction. Becauset here are more active sites available to change substrate into product.
  151. 151. K1 E+S K2 ES K -1 E+S The formation of product is essentially irreversible the severe reaction does not occur to any appreciable retent. ES E+P K -2
  152. 152.  Thus K-2 is much less than K2. if the product is removed the reaction will be complete, but if not removed the reaction will remain incomplete. Under steady-state conditions, the net effect of enzyme is to convert substrate products as rapidly as the products are removed. pH dependence of enzyme activity is result of several effects.
  153. 153.  Ionization gps. In the active site of the enzyme in the substrate or in enzymesubstrate complex can affect catalysis depending or whether the gps. Are dissociated or undissociated. Ionization of these gps. Depends on their pK values, the chemical properties of surrounding gps. & the pH of the reaction medium .
  154. 154. Changes in pH effect the binding of the substrate at the active site of enzyme & also the rate of breakdown of ES complex e.g. catalytic activity may require an amino gps of the enzyme be in protonated from (-NH3+) at alkaline pH this gp is deprotonated & the rate of reaction therefore declines. The enzymes in living systems function at nearly constant pH because they are in an environment that contains buffers.
  155. 155.  The pH enzyme activity profile of most enzymes delineates or bell shaped curve each exhibiting an optimal pH- ie the pH at with enzyme activity is maximal. E.g. pepsin, a digestive enzyme in the stomach is maximally active at pH2. whereas other enzymes arte designed to work at neutral pH. Are denatured by such an acidic ….
  156. 156. Trypsin Alk. Po4ase (Vo ) reaction velocity Pepsin 3 5 7 9 11
  157. 157. Extremes of pH can also lead to denaturation of the enzyme ΅ the structure of catalytically active protein molecule depends on the ionic character of the amino acid side chain.
  158. 158. Increased velocity with temperature: The reaction velocity ↑es with temperature un till a peak velocity is reached. This ↑es the result of the ↑sed no. of molecules having sufficient energy to pass over the energy barrier & form the products of reaction.
  159. 159. Further elevation of temperature results in ↑es in the reaction velocity as a result of temperature induced denaturation of enzyme.
  160. 160. The rate at which the substrate changes to products is directly proportional to time.
  161. 161. Many enzymes require the presence of metal ions to function. Those enzymes that bind the metal ions loosely are called metal-activated enzymes. Common … activators include Mg+2, Mn +2, fe2+, Ca2+, Zn2+, K+ aminos may also function as activators. Magnesium is an obligate activator for allkinase enzyme i.e PO4 transfer enzymes. Amylase is a calcium metallo enzyme that displays full activity in the presence of variety inorganic ions(Ce`, Br`, NO-+
  162. 162.  In activation dependant enzyme reactions the substrate should be present in … concentration. Excess activator may also function to inhibit activity. Therefore in some cases optimal activator should be used. So in absence of coenzyme of activator enzyme may be inactive or sluggish.
  163. 163. Any substance that can dimish the velocity of an enzyme catalysed reaction is called as inhibitor. The inhibitors are:A. Reversible inhibitors B. irreversible inhibitors  i. ii. iii. Competetive inhibitor …. ….
  164. 164. Competative inhibitors - ↑se Km but has no effect on V max Now competetive inhibitors - ↓ both Vmax + Km.
  165. 165.      Providing information about shape of active site Types of αα side chains there Working out enzyme mechanism Providing information about control of metabolic pathway Design of drugs.
  166. 166. Reversible inhibitors bind to enzyme through noncovalent bonds. Dilution of the enzyme inhibitor complex results in dissociation of reversibility bond inhibitor & recovery of enzyme activity. Its further types are:
  167. 167. In reversible competetive inhibition, inhibitors compete with the substrate for binding to active site & they form enzyme inhibitor complex.
  168. 168. The effect of a competitive inhibitor is reversed by ↑ing [S] . At a sufficiently high [S] the reaction velocity reaches the Vmax observed in the absence of inhibitor.
  169. 169. In the presence of competitive inhibitor more substrate is needed to achieve ½ Vmax.
  170. 170. Reaction Velocity V max V max 2 Km Km [S]
  171. 171. I Vo Comp. inhibition No inhibition I V max -I Km -I Km I [ -S]
  172. 172. Apparent competitive inhibition occurs in 4 different circumstances.
  173. 173. Compete with substrate for binding at active site e.g. inhibition of succinate dehydrogenase COOHI CH2 I CH2 I COO Succinate Succinate dehydregenase + FAD H - COOI C II C + I COO- - H FADH2
  174. 174. In this reaction FAD, a coenzyme serves as a hydrogen acceptor. This enzyme is competeively inhibited by malonate, oxalate or oxaloocetate, all are structural analogues of succinate.
  175. 175. COOI CH2 I COOMalonate COOI COOOxalate COOI C =O I CH2 I COOOxalocetate
  176. 176. Competitive inhibition of a biosynthesis step in folate synthesis accounts for the antimicrobial action of sulforaminades which are structural analogues of para amino benzoic acid (PABA)
  177. 177. O II - H2N C - OH PABA H2N O II - Sulfonamide C - NH2
  178. 178. Para amino benzoic acid is used by the bacteria in the synthesis of folic acid. Sulforamides inhibit the bacterial enzymes responsible for incorporation of PABA into 7-8 dihydropteroic acid & lead to inhibition of growth of a wide range of gram+ve & gram –ve microorganism.
  179. 179. Microorganism susceptable to sulfonamides are those with synthesize their … folic acid derived from host. Sulfonamides however have no effect on host cells. That require preformed folic acid.
  180. 180. Uric acid is the end product of purine catabolism in humans. Hypoxanthine oxidase xanthine uric Xanthine oxidase Xanthine acid allopurinol a structural analogue of hypoxanthine is a comprtetive inhibitor as well as substrate for xanthine oxidase. 
  181. 181. OH OH N N N N HYPOXANTHINE N I H N N ALLOPURINOL Allopurine inhibits the formation of xanthine & of uric acid. N I H
  182. 182. In two substrate enzyme-catalysed reactions, high [] of 2nd substrate may complete with the first substrate for binding e.g. reaction catalysed by aspartate aminotransferase. L-aspartate & ketoglutarate Lglutamate + oxaloacetate.
  183. 183.  Competetive inhibition in reversible reactions due to accummlation of products. Inhibits e.g. alkaline phosphatase causes hydrolysis of a wide variety of organic mono-phosphate esteers x the corresponding alcohols & in organic PO4 occurs, the inorganic Po4 acts as a competetive inhibitor. Both the inhibitor & the substrate have similar binding affinutis.
  184. 184.   Metal ions act as inhibitors:- In reactions that require metal ions as cofactors. similar,metal ions can compete for the same binding site on enzyme e.g. prymate kinase catalyzees the reaction. Phosphoand pyrumate & ADP→ ATP + pyrmate for which K+ is an obligatory activators whereas Na+ + Li+ are potent competetive inhibitors.
  185. 185.    Inhibitor does not usually bear any structural resemblance to the substrate & it binds to the enzyme at site distinct from substrate b/w the inhibitor & substrate & inhibition cannot be overcome by ↑se of substrate concentration. An inhibitor may bind either to a free enzyme or to an enzyme-substrate complex in both cases, the complex is catalytically in active E+I E I (inactive) ES + I ESI ( inactive)
  186. 186. Vmax is reduced as non-competetive inhibition cannot be overcome by ↑ing the concentration of substrate.
  187. 187. Km is un affacted because the affinity of S for E is unchanged.
  188. 188.  Lead covalent bounds with sulphydeyl group side of cyteine in proteins e.g. ferochelatase an enzyme that catalyze the insertion of Fe+2 into porphyrin & δ amino lenulinate dehydrase, both enzymes are sensitive to inhibition by lead i.e why lead poisoning cause anemia.
  189. 189. For activity are inhibited by chelating agent e.g ethylenediamine tetra acetate that remove the metal ion from enzyme.
  190. 190.  Uncompetitive inhibitions combine reversibly only with ES to form ESI which cannot yield product. It is not reversed by ↑ substrate concentration. ES + I ESI Inhibitions bind only to the ES at a site distiunct from active site.
  191. 191. Uncompetitive inhibition is rarely observed in single-substrate reaction. It is more common in 2-substrate reactions with a double displacements reaction mechanism e.g. inhibition of intestinal alkaline PO4 by L-phynylalanine.
  192. 192.  Occurs when the inhibition acts at or near the active site of the enzyme with covalent modification of the active site or when the inhibitor binds so tightly that there is no dissociation of enzyme inhibitor. Thus physical separative processes are ineffective in removing the irreversible inhibitor from the enzyme. E + I → EI
  193. 193. e.g. i. Enzymes that contain free sulphydeyl groups at the active site e.g. glyceraldelyde -3-Po4 dehydrogenase. Enzyme – SH+ iodoaaxetic acid → inactive covalent derinactive of enzyme
  194. 194. ii. Enzymes with seryl hydroxyl group at active site. These enzymes can be inactiveated by organophosphorus compounds. Several organophosphorous compounds are used as agricultural insecticides, improper exposure to which can result in toxic manifestation & death.
  195. 195.  Acetylcholine is a neurotransmitter which is related on arrival of a nerve impulse at the ending of neuron & it ↑ses the premeability of Na+ across the postsyoptic membrane & result in progation of action potential. Acetycholine is quickly destroed by acetylcholinestrase.
  196. 196.  The organophosphorous compounds cause inactivation of acetylcholine esterase, the continued presence of acetylcholine causes extended transmission of nerve impulses. In muscle fibers continues depolarization leads to paralysis. The cause of death is respiratory failure due to poralysis of respiratory muscles.
  197. 197. Competitive inhibition is the basis for the treatment of some intoxicants e.g. nethanol which is widely used in industry as a solvent. Methanol is metabolised mainly in liver and kidneys.
  198. 198. Alcohol dehydrogenase Methanol ↓ formaldelyde → inhibited by ethanol formic acid
  199. 199.  Major toxic effects are caused by formaldehyde causing damage to retinal cells leading to blindness. Formic acid – severe acidosis – deaths . Retardation of 1st step is accomplished by administration of ethanol, the oxidation products of which are not as toxic as those of methanol.
  200. 200. Drugs can also inhibit enzymes e.g. Penicillin which inhibits the reaction with transpeptidase that is important in the development of bacterial membranes. Thus destroying normal growth of bacteria.
  201. 201. Sr. inhibitor no. 1 aspirin 2 Allopurinol 3 SFluorowaci d Penicillin 4 Target enzyme Cyclo oxygenase Xanthine oxidase Thymidylate synthesis transpeptidase Effect on application Antiinflammatory T/m of gout Anti cancer Antibacterial