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Enzymes, Structure, Classification and Mechanism Dr.Kamlesh shah, PSSHDA, KADI
- 1. ENZYMES Dr.Kamlesh shah A protein with catalytic properties due to its power of specific activation
- 2. Enzyme Fundamentals • Enzymes are protein complexes that speed up biochemical reactions by lowering the activation energy • Enzymes accelerate reactions by facilitating the formation of the transition state • The position of the equilibrium, enthalpy of reaction, and free energy of the reaction are unchanged by an enzyme • The enzymes themselves are the same after the reaction as they was before • Enzymes are powerful and highly specific catalysts • Free energy is a useful thermodynamic function for understanding enzymes • The Michaelis-Menten model accounts for the kinetic properties of many enzymes • Enzymes can be inhibited by specific molecules • Vitamins are often precursors to coenzymes
- 3. Some Enzyme Terminology • Enzyme – a biomolecule that catalyzes biochemical reaction by lowering activation energy • Substrate – the substance that undergoes a chemical change by an enzyme • Absolute Specificity – the characteristic that an enzyme acts on only one substrate • Relative Specificity – the characteristic that an enzyme acts on several structurally related substrates • Stereochemical Specificity – an enzyme's ability to distinguish between stereoisomers • Cofactor – a nonprotein molecule or ion required by an enzyme for catalytic activity • Coenzyme – an organic molecule required by an enzyme for catalytic activity
- 4. More Enzyme Terminology • Apoenzyme – a catalytically inactive protein formed by removal of the cofactor from an active enzyme • Active Site – the location on an enzyme where a substrate is bound and catalysis occurs • Enzyme Activity – the rate at which an enzyme catalyzes a reaction • Turnover Number – the number of molecules of substrate acted upon by one molecule of enzyme per minute • Enzyme International Unit (IU) – a quantity of enzyme that catalyzes the conversion of 1 micromole of substrate per minute under specified conditions • Optimum Temperature – the temperature at which enzyme activity is highest
- 5. And More Enzyme Terminology • Optimum pH - the pH at which enzyme activity is highest • Extremozyme – an enzyme that thrive in extreme environments • Enzyme Inhibitor – a substance that decreases the activity of an enzyme • Competitive Inhibitor – an inhibitor that binds to the active site of an enzyme • Noncompetitive Inhibitor – an inhibitor that binds at a location other than the enzyme’s active site • Zymogen (proenzyme) – the inactive enzyme precursor • Modulator – a substance that binds to an enzyme at a location other than the active site that alters the enzyme's catalytic activity
- 6. And Yet More Enzyme Terminology • Allosteric Enzyme – an enzyme with a quaternary structure whose activity is changes by the binding of a modulator • Activator – a substance that binds to the allosteric enzyme and increases its activity • Feedback Inhibition – a process in which the end product of a sequence of enzyme catalyzed reaction inhibits an earlier step in the process • Enzyme Induction – the synthesis of enzyme in response to a cellular need • Isoenzyme – a slightly different form of the same enzyme produced by different tissues • Holoenzyme – apoenzyme + cofactor
- 7. Two Fundamental conditions for life Ability to self replicate. Ability to catalyze chemical reactions efficiently and specifically. Example
- 8. Enzymes Reaction catalysts of biological system. Extraordinary catalytic power. High degree of specificity. Function in aqueous solutions under mild conditions of temperature and pH.
- 9. Practical Importance Inheritable genetic disorders (deficiency or total absence of enzymes). Excessive activity of enzymes may cause diseases. Diagnosing certain illness. Many drugs exert their effect by interacting with enzymes. Important practical tools in medicine, chemical industry, food processing and agriculture.
- 10. History 1850 – Louis Pasteur Concluded that fermentation of sugar to alcohol by yeast is catalyzed by “ferments”. 1878 – The molecules extracted from cells responsible for catalysis were named “Enzymes” by Frederick Kuhne. 1897 – Eduard Buchner Disproved “Vitalism” and stated that fermentation could be carried out by yeast extracts.
- 11. Contd…. 1926 James Sumner Isolated and crystallized Urease enzyme and postulated that “ All Enzymes are Proteins”. John Northrop and Moses Kunitz crystallized pepsin, trypsin and other digestive enzymes.
- 12. Enzyme structure All Enzymes are Proteins Exception – Catalytic RNA molécules Molecular Weight – 12,000 to >1 million. They have a globular shape. A complex 3-D structure. 1o,2o,3o, and 4o structures of protein enzymes are essential for their Catalytic activity. Human pancreatic amylase
- 13. Enzymes Endoenzymes or Intracellular Act within the cells in which they are produced Metabolic enzymes Plant enzymes Exoenzymes or extracellular Liberated by living cells and act outside in its environment Chiefly act as digestive enzymes Microbial enzymes
- 14. Cofactors An additional non-protein molecule that is needed by some enzymes to help the reaction. Example: • Inorganic ions such as Fe2+, Mg2+, Mn2+, or Zn2+ • Complex organic or metalloorganic molecule called a Coenzyme. Some enzymes require both Coenzymes as well as one or more metal ions for their activity. Nitrogenase enzyme with Fe, Mo and ADP cofactors
- 15. Cofactors….. Tightly bound cofactors are called Prosthetic groups. Cofactors that are bound and released easily are called coenzymes Many vitamins are coenzymes. Complete catalytically active enzyme together with bound coenzyme/ metal ion – Holoenzyme. Protein part of the enzyme - Apoenzyme or Apoprotein.
- 16. The active site One part of an enzyme, the active site, is particularly important The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily
- 19. Nomenclature Enzyme commission - Six classes – 1st Main class, 2nd Sub class, 3rd Sub subclass – 4th molecule 1.Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases Eg: Histidine carboxylyase: EC. 4.1.1.22 Lyase C - C C-COOH Histidine
- 20. Chemical reactions Chemical reactions need an initial input of energy = THE ACTIVATION ENERGY During this part of the reaction the molecules are said to be in a transition state.
- 21. Reaction pathway
- 22. An enzyme controlled pathway Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions.
- 23. Making reactions go faster Increasing the temperature make molecules move faster Biological systems are very sensitive to temperature changes. Enzymes can increase the rate of reactions without increasing the temperature. They do this by lowering the activation energy. They create a new reaction pathway “a short cut”
- 24. The substrate The substrate of an enzyme are the reactants that are activated by the enzyme Enzymes are specific to their substrates The specificity is determined by the active site
- 25. The Lock and Key Hypothesis Fit between the substrate and the active site of the enzyme is exact Like a key fits into a lock very precisely The key is analogous to the enzyme and the substrate analogous to the lock. Temporary structure called the enzyme-substrate complex formed Products have a different shape from the substrate Once formed, they are released from the active site Leaving it free to become attached to another substrate
- 26. The Lock and Key Hypothesis Enzyme may be used again Enzyme- substrate complex E S P E E P Reaction coordinate
- 27. The Lock and Key Hypothesis This explains enzyme specificity This explains the loss of activity when enzymes denature
- 28. The Induced Fit Hypothesis Some proteins can change their shape (conformation) When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation The active site is then moulded into a precise conformation Making the chemical environment suitable for the reaction The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)
- 29. The Induced Fit Hypothesis This explains the enzymes that can react with a range of substrates of similar types Hexokinase (a) without (b) with glucose substrate http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html
- 30. Factors affecting Enzymes substrate concentration pH temperature inhibitors
- 31. Substrate concentration: Non-enzymic reactions The increase in velocity is proportional to the substrate concentration Reaction velocity Substrate concentration
- 32. Substrate concentration: Enzymic reactions Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. If you alter the concentration of the enzyme then Vmax will change too. Reaction velocity Substrate concentration Vmax
- 33. The effect of pH Optimum pH values Enzyme activity Trypsin Pepsin pH 1 3 5 7 9 11
- 34. The effect of pH Extreme pH levels will produce denaturation The structure of the enzyme is changed The active site is distorted and the substrate molecules will no longer fit in it At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur This change in ionisation will affect the binding of the substrate with the active site.
- 35. The effect of temperature Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in temperature. For chemical reactions the Q10 = 2 to 3 (the rate of the reaction doubles or triples with every 10°C rise in temperature) Enzyme-controlled reactions follow this rule as they are chemical reactions BUT at high temperatures proteins denature The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation.
- 36. The effect of temperature Temperature / °C Enzyme activity 0 10 20 30 40 50 Q10 Denaturation
- 37. The effect of temperature For most enzymes the optimum temperature is about 30°C Many are a lot lower, cold water fish will die at 30°C because their enzymes denature A few bacteria have enzymes that can withstand very high temperatures up to 100°C Most enzymes however are fully denatured at 70°C
- 38. Inhibitors Inhibitors are chemicals that reduce the rate of enzymic reactions. The are usually specific and they work at low concentrations. They block the enzyme but they do not usually destroy it. Many drugs and poisons are inhibitors of enzymes in the nervous system.
- 39. The effect of enzyme inhibition Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly. Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase.
- 40. The effect of enzyme inhibition Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. There are two categories.
- 41. The effect of enzyme inhibition 1. Competitive: These compete with the substrate molecules for the active site. The inhibitor’s action is proportional to its concentration. Resembles the substrate’s structure closely. Enzyme inhibitor complex Reversible reaction E + I EI
- 42. The effect of enzyme inhibition Succinate Fumarate + 2H++ 2e- Succinate dehydrogenase CH2COOH CH2COOH CHCOOH CHCOOH COOH COOH CH2 Malonate
- 43. The effect of enzyme inhibition 2. Non-competitive: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site. Examples Cyanide combines with the Iron in the enzymes cytochrome oxidase. Heavy metals, Ag or Hg, combine with –SH groups. These can be removed by using a chelating agent such as EDTA.
- 44. Applications of inhibitors Negative feedback: end point or end product inhibition Poisons snake bite, plant alkaloids and nerve gases. Medicine antibiotics, sulphonamides, sedatives and stimulants
- 45. Enzymes in medicine Glucose oxidase Glucose Hydrogen peroxide peroxidase Dye: Blue---Green---Brown Dye changes according to amount of glucose Enzyme-linked immunosorbent assays (ELISAs) detect antibodies to infections. Glucose oxidase + peroxidase + blue dye on dipsticks to detect glucose in urine:
- 46. Fermentation Culture supernatant Centrifugation to remove cells Liquid preparation to animal feed market Fermentation Culture supernatant Fermentation Cell pellet Intracellular fraction Animal feed enzyme Analytical enzyme Therapeutic protein Centrifugation to remove cells Centrifugation to remove medium Protein precipitation Cell lysis Centrifugation Protein fraction Protein precipitation Protein fraction 1 or 2 purification steps Semi-purified protein 3-4 purification steps Homogeneous protein Sterile bottling To pharmaceuticals market Lyophilisation Bottling To chemicals market
- 47. Fungal Enzymes Enzyme EC Sources Location Application a-Amylase 3.2.1.1 Aspergillus E Baking Catalase 1.11.1.6 Aspergillus I Food Cellulase 3.2.1.4 Trichoderma E Waste Dextranase 3.2.1.11 Penicillium E Food Glucose oxidase 1.1.3.4 Aspergillus I Food Lactase 3.2.1.23 Aspergillus E Dairy Lipase 3.1.1.3 Rhizopus E Food Rennet 3.4.23.6 Mucor miehei E Cheese Pectinase 3.2.1.15 Aspergillus E Drinks Protease 3.4.23.6 Aspergillus E Baking E: extracellular enzyme; I: intracellular enzyme
- 48. Bacterial Enzymes Enzyme Sources Application a-Amylase 3.2.1.1 Bacillus E Starch b-Amylase 3.2.1.2 Bacillus E Starch Asparaginas e 3.5.1.1 Escherichia coli I Health Glucose isomerase 5.3.1.5 Bacillus I Fructose syrup Penicillin amidase 3.5.1.11 Bacillus I Pharmac eutical Protease 3.4.21.14 Bacillus E Detergent
- 49. Amylases • hydrolysis of starch (glucose polymer), one of the most readily available plant polysaccharides • production of sweeteners from starch: maltose or glucose syrups (further transformation to high fructose syrup with glucose isomerase) • starch hydrolyates used as additives in the manufacture of candies, baked goods, canned goods, and frozen foods Proteases • used in laundry detergents
- 50. Glucose Isomerase D-glucose ketoisomerase: causes the isomerization of glucose to fructose since reaction is reversible the rtion of glucose and fructose depends on the enzyme and reaction conditions high fructose corn syrup fructose 2x sweeter than sucrose Chymosin site-specific proteolysis by chymosin detaches hydrophilic “tails” of κ- casein resulting in coagulation (curlding) calf chymosin (prochymosin) cloned and expressed in E. coli (first genetically engineered protein approved for human consumption, 1990)
- 51. Aspartic acid and phenylalanine are synthesized into aspartame the artificial sweetener through enzyme Thermolysin. Aspartame is a non-nutritive sweetener of diet soft drinks and other foods sold as low- calorie or sugar-free products.
- 52. Microbial enzymes and their Applications