Bài giảng Basic Food Chemistry - Chapter 3: Enzyme

Nội dung text: Bài giảng Basic Food Chemistry - Chapter 3: Enzyme

1. CONTENTS ❑Nomenclature and Classification ❑Mechanism of enzyme action ❑Catalytic mechanism ❑Enzyme activity ❑Factors influence on enzyme activity ❑Enzyme Kinetics ❑Enzyme production 12/31/2022 2
2. ENZYME NOMENCLATURE ❑ Common name Substrate/ activity + ase → Examples: ▪ urease catalyzes hydrolysis of urea ▪ DNA polymerase catalyzes the polymerization of nucleotides to form DNA 12/31/2022 4
3. ENZYME NOMENCLATURE ❑ EC (Enzyme Commission) systematic name: → [International Union of Biochemistry and Molecular Biology: IUBMB] → Systematic name indicating: ▪ The substance acted on ▪ The functional group acted on ▪ The type of reaction catalyzed → All EC names end in –ase 12/31/2022 6
4. EC CLASSIFICATION OF ENZYMES Enzymes: grouped into 6 major classes (based on the rxns which they catalyze) 12/31/2022 8
5. ENZYME CLASSIFICATION 12/31/2022 10
6. Lyases, Isomerases and Ligases
7. Lyases ❑ Lyases: cleaving bonds ▪ Ex: aldolases: fructose diphosphate aldolase: in glycolysis) ▪ Thiolases: β-ketoacyl-CoA thiolase: breakdown of fatty acids 12/31/2022 by TDLV 14
8. Ligases ❑ Ligases: catalyzing synthesis of bonds 12/31/2022 by TDLV 16
9. CLASSWORK ?: Predict the substrates for the following enzymes: a) Maltase b) Peptidase c) Glucose 6-phosphate isomerase 12/31/2022 18
10. STRUCTURE OF AN ENZYME ▪ Some enzymes are proteins ▪ Some enzyme are RNAs ▪ Some contains both a protein (apoprotein/apoenzyme) and a nonprotein → Nonprotein: either a coenzyme (vitamins) or a cofactor (inorganic ions) → Some enzymes require both a coenzyme and one cofactor for activity 12/31/2022 20
11. COENZYME Table2: Some common coenzymes 12/31/2022 22
12. LOCK-AND-KEY THEORY ❑ The lock-and-key theory: → explains the high specificity of enzyme → Enzyme surface accommodates substrates having specific shapes and sizes → Only specific substances “fit” in an active site to form an ES complex. 12/31/2022 24
13. Fig. : lock-and-key theory Fig. : induced-fit theory 12/31/2022 26
14. MECHANISM OF ENZYME ACTION ❑Step 2: form enzyme-product complex ▪ Enzyme-product complex formed ES EP 12/31/2022 28
15. MECHANISM OF ENZYME ACTION 12/31/2022 30
16. ENZYME CATALYSIS MECHANISM 1. Acid-base catalysis: hydrolysis of ester/ peptide bonds, phosphate group reactions, addition to carbonyl groups, etc. 2. Covalent catalysis: formation of a catalyst- substrate covalent bond Serine proteases: acyl-serine intermediate Cysteine proteases: acyl-cystein intermediate Protein kinases and phosphatases: phospho-amino acid intermediates 12/31/2022 by TDLV 32
17. ▪ Covalent catalysis: a transient covalent bond is formed between the enzyme and the substrate 12/31/2022 by TDLV 34
18. Mechanisms of catalysis 12/31/2022 by TDLV 36
19. Mechanisms of catalysis Metal ion catalysis: • Metal ions often used for one or more of the following: – binding substrates in the proper orientation (e.g. Cytochromes) – mediating oxidation-reduction reactions – electrostatically stabilizing or shielding negative charges that would otherwise repel the attack from an electrophile (electrostatic catalysis) – Simply stabilize the catalytically active form of the enzyme 12/31/2022 by TDLV 38
20. Mechanisms of catalysis K+ • The most abundant intracellular cation known to activate many enzyme • Its role is largely to bind the (-)ly charged groups in NZ → more active form Mg+2 and Ca+2 • Mg+2 (intracellular NZs) and Ca+2 (extracellular NZs) • Ca+2 → extracellular NZs, e.g. Salivary and pancreatic - amylase: maintain the structure required for activity • Mg+2→ intracellular NZs, most kinases 12/31/2022 by TDLV 40
21. Mechanisms of catalysis 4. Catalysis by alignment Conformational distortion (Transition state) (of substrate) • "Strain“: binding of the substrate to the enzyme caused the substrate to become distorted toward the transition state • Transition state stabilization: the transition state makes better contacts with the enzyme than the substrate does. 12/31/2022 by TDLV 42
22. ENZYME ACTIVITY ❑ Enzyme activity: the total units of enzyme activity in a solution ❑ Specific activity: the number of enzyme activity units per milligram of total protein → Specific activity: a measure of enzyme purity → the higher the specific activity, the purer the enzyme 12/31/2022 44
23. ❑ Enzyme assays: → experiments performed to measure enzyme activity → often done by monitoring the rate at which a characteristic color of a product forms or the color of a substrate decreases → colorimetric method → For reactions involving H+ ions, monitoring the rate of change in pH over time 12/31/2022 46
24. ACTIVATION OF ZYMOGEN ❑ Zymogens (proenzymes): inactive precursors of an enzyme ▪in their active form → degrade the internal structures of the cell → are synthesized and stored as inactive precursors ▪when the enzyme is needed →the zymogen is released and activated (cleavage of one or more peptide bonds) ▪digestive enzymes: pepsin, trypsin, chymotrypsin 12/31/2022 48
25. ALLOSTERIC REGULATION ❑ Modulators: compounds alter enzymes by changing the 3D conformation of the enzyme → activators: increase enzyme activity → inhibitors: decrease enzyme activity: non-competitive inhibitors • Allosteric enzymes: enzymes with quaternary structures with binding sites for modulators 12/31/2022 50
26. GENETIC CONTROL ❑ Synthesis of enzymes is under genetic control by nucleic acids → Increasing the number of enzymes molecules present through genetic mechanisms → increase production of needed products • Enzyme induction: enzymes are synthesized in response to cell need → allows an organism to adapt to environmental changes 12/31/2022 52
27. Enzyme Activity Enzyme activity: total units of activity in a given volume of solution. Specific activity: the number of units of activity per amount of total protein (mass) → follow the increasing purity of an enzyme through several procedural steps. Molecular activity: Used to compare activities of different enzymes. Also called the turn-over number (TON = kcat)
28. Enzyme Activity New international units: Unit of enzyme activity: mol substrate transformed/sec = katal Specific activity: (mol substrate transformed/sec)/kg E = katal/kg E Molecular activity: (mol substrate transformed/sec)/mol E = katal/mol E
29. REGULATION OF ENZYME ACTIVITY ❑Enzymes are often regulated by the cell ❑Cells use several methods to control when & how well enzymes work 12/31/2022 by TDLV 58
30. THE EFFECT OF TEMPERATURE ❑ Increasing temperature → reaction rate increases • Because enzymes are proteins, beyond a certain temperature, the enzyme denatures. • Every enzyme has an optimum temperature (at which the enzyme activity is highest) → above or below the optimum temperature → the rate is lower 12/31/2022 60
31. THE EFFECT OF pH VALUE 12/31/2022 62
32. THE EFFECT OF ENZYME AMOUNT • Enzyme amount increases → reaction rate increases (at constant substrate concentration) • Linear relationship between reaction rate and enzyme amount Enzyme amount: too high → no effect on rxn rate (substrate concentration becomes the limiting factor) 12/31/2022 64
33. ENZYME SPECIFICITY • Enzymes have varying degrees of specificity for substrates • Enzymes specificity - a single substrate (absolute) - a group of similar substrates - a particular type of bond - Stereochemical specificity: Only work with the proper D- or L- form 12/31/2022 by TDLV 66
34. Enzyme kinetics 12/31/2022 by TDLV 68
35. Michaelis – Menten equation 12/31/2022 by TDLV 70
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41. Kcat: turnover number (TON) 12/31/2022 by TDLV 82
42. Vo = Vmax[S] can be transformed by Km + [S] taking reciprocals. → plot the curve as a straight line in the form y = mx + c
43. A Lineweaver Burk plot (double reciprocal plot) ❖y = mx + b • y = 1/vo, m (slope) = Km/Vmax, x = 1/[S] • y-intercept, b = 1/Vmax 12/31/2022 by TDLV 86
44. Kcat Kcat: more general rate constant: to describe the limiting rate of any enzyme-catalyzed reaction at saturation. If the reaction has several steps and one is clearly rate-limiting→ kcat is equivalent to the rate constant for that limiting step 12/31/2022 by TDLV 88
45. Kcat 12/31/2022 by TDLV 90
46. kcat/Km – chymotrypsin specificity: active site best accommodates substrates with a bulky hydrophobic or aromatic residue contributing carbonyl group to peptide bond to be hydrolyzed. 12/31/2022 by TDLV 92
47. Calculation of kinetic parameters Example 1 The rate of an enzyme catalyzed reaction is 35 -4 -5 μmol/min at [S] = 10 M, (KM = 2 x 10 ). Calculate the velocity at [S] = 2 x 10-6 M.
48. Example 3 Ten micrograms of carbonic anhydrase (MW = 30000) in the presence of excess substrate exhibits a reaction rate of 6.82 x 103 μmol/min. At [S] = 0.012 M the rate is 3.41 x 103 μmol/min. a. What is Vm ? b. What is KM ? c. What is k2 (kcat) ?
49. EXAMPLE 5: Determining [S] In a separate richase experiment using [Et]=10 nM, -1 the reaction velocity, V0, is measured as 3 μMs . What is the [S] used in this experiment? 12/31/2022 by TDLV 98
50. INHIBITION ❑ Enzyme inhibitor: a substance that decreases the rate of an enzyme-catalyzed reaction – poisons, medicines inhibit one or more enzymes → decrease the rate of the enzyme-catalyzed reactions –Irreversible inhibition: when an inhibitor forms a covalent bond with a specific functional group of an enzyme → inactivating enzyme 12/31/2022 100
51. IRREVERSIBLE INHIBITION → Heavy metal poisoning: when mercury or lead ions bind to —SH groups on enzymes → Heavy metals also cause protein denaturation → Pb and Hg cause permanent neurological damage 12/31/2022 102
52. REVERSIBLE INHIBITION ❑ Reversible inhibitor: binds reversibly to an enzyme → establishing an equilibrium between the bound and unbound inhibitors: – inhibitor combines with enzyme → active site blocked → no further catalysis takes place → inhibitor can be removed from the enzyme by shifting the equilibrium 12/31/2022 104
53. . COMPETITIVE INHIBITION E + S ES E + P + Enzyme I S I EI Inhibitor binds only to the free enzyme, not to the ES Inhibitor competes complex with substrate for the same binding site 12/31/2022 by TDLV 106
54. In the presence of a competitive inhibitor, the Michaelis- Menten equation becomes The Km observed in the presence of the competitive inhibitor, called the “apparent” Km= αKm, α can be calculated from the change in slope at any given [I]. Knowing [I] and α → calculate KI from the expression12/31/2022 by TDLV 108
55. • Km increases with increasing [Inhibitor] (more inhibitor occupies the active site → effectively changing the affinity of the active site for the substrate). • As [S] increases, Vo ➔ Vmax (if [S] very large compared with [In] → S outcompetes In.
56. • NON-COMPETITIVE INHIBITION (NCI)
57. Note the differences between NCI and CI: In NCI, the Km remains the same in the presence of the inhibitor but the Vmax decreases. The new Vmax is called Vmax apparent.
58. In the presence of an uncompetitive inhibitor, the Michaelis-Menten equation is altered to: 12/31/2022 by TDLV 118
59. For uncompetitive and mixed inhibition, changes in axis intercepts signal changes in Vmax and Km 12/31/2022 by TDLV 120
60. 12/31/2022 by TDLV 122
61. MIXED INHIBITION 12/31/2022 by TDLV 124
62. . IRREVERSIBLE INHIBITION MECHANISM Enzyme Inhibitor binds to the enzyme S irreversibly through formation O I of a covalent bond with the enzyme , permanently inactivating the enzyme E + S ES E + P + I Vmax decreases No effect on Km EI 12/31/2022 by TDLV 126
63. Allosteric Enzymes Allosteric enzymes have one or more sites in addition to the active site. These allosteric sites bind effector molecules which can modify the rate of catalysis.
64. Properties of Allosteric enzymes 1. Catalyze essentially irreversible reactions; are rate limiting 2. Generally contain more than one polypeptide chain 3. Do not follow Michaelis-Menten Kinetics 4. Are regulated by allosteric activators or inhibitors 5. Can be up-regulated by allosteric activators at constant [S] 6. Can be down regulated by allosteric inhibitors at constant [S] 7. Activators and Inhibitors need not have any structural resemblance to substrate structure 12/31/2022 by TDLV 130
65. To calculate the rate of reaction, plot a graph of the extent of reaction ( either formation of products or disappearance of reactants) against time. → Reaction-Time Plot or Progress Curve.
66. → more useful to express enzyme rate not as initial rate but as specific activity. Specific activity = μmol substrate converted per minute per mg protein. = μmol min-1mg-1 → give information on purity of enzyme: The higher the specific activity, the greater the purity
67. Sequential Reaction • Ordered sequential • Random sequential 12/31/2022 by TDLV 138
68. Initial Velocity Plots Ping-pong reaction Sequential reaction 12/31/2022 by TDLV 140
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70. PROENZYMES (ZYMOGENS) ❑ Enzymes manufactured in inactive form ❑ Activated when small part of polypeptide chain removed Trypsinogen enteropeptidase Trypsin Chymotrypsinogen Trypsin Chymotrypsin procarboxypeptidase Trypsin Carboxypeptidase Digestive Proteases Enzymes Cleave peptides 12/31/2022 by TDLV 144
71. Enzymes in Industry • Feed enzymes • Enzymes to assist in the digestibility of animal feeds (cellulase, xylanase, phytase) • Waste management • Lipases as drain-cleaning agents • Diagnostic enzymes • Reporter enzymes (alkaline phosphatase, glucose oxidase, b-glucosidase) and diagnostic enzymes (DNA polymerase) 12/31/2022 by TDLV 146
72. APPLICATIONS OF ENZYMES IN FOOD TECH • Cheese making (rennet) • Meat tenderization (papain, trypsin) • Brewing (trypsin, pepsin) • Improve flavor, quality, or appearance 12/31/2022 by TDLV 148
73. Enzymatic Browning • Reaction of oxygen and the enzyme PPO (polyphenol oxidase) (phenolase) → desirable and undesirable color and structure changes (Desirable changes – browning of raisins, , Undesirable changes fruit becomes discolored, mushy, bruised) 12/31/2022 by TDLV 150
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75. Immobilising methods Method Description Adsorption Enzyme mixed with immobilising supports e.g. porous carbon, glass beads, clay & resins with hydrophobic interactions and ionic links*. Detachment is possible due to weak bonds but reaction rates are high if active site is displayed. Covalent Bonding Enzymes covalently linked to insoluble material e.g. clay using cross linking agent (gluteraldehyde/sepharose) Binding is strong, so very little enzyme leakage, but small quantities only Entrapment Enzymes trapped in a gel bead or cellulose fibre network. Active sites are not affected, but reaction rates reduced if substrate can’t get through trapping barrier Membrane Enzymes separated by a partially permeable membrane. Enzyme Separation on one side, substrate on another. Substrate molecules and products can pass across the membrane. *An ionic bond is a type of chemical bond formed through electrostatic attraction between two oppositely charged ions. 12/31/2022 by TDLV 154
76. Enzymes are covalently bonded to a matrix such as cellulose or collagen Another more expensive method involves enzymes which are both covalently bonded to, and cross-linked within, a matrix Cross-linking and covalent bonding may cause some enzymes to lose their catalytic activity especially if the active site is involved in forming the linkages
77. Disadvantages of Immobilised enzymes • Additional time, equipment and materials needed → expensive • Can be less active (they do not mix freely with the substrate • Contamination can be occurred 12/31/2022 by TDLV 158
78. METHODS OF IMMOBILISATION 2. Adsorption • Enzyme attached by weak physical forces to a support matrix (glass beads or carbon particles) • Does not chemically modify enzyme →adsorption process may cause enzymes to loose their shape → activity loss • Molecules may become detached during the bioconversion reaction 12/31/2022 by TDLV 160
79. Task 1. Outline the advantages and disadvantages of immobilised enzymes 2. Explain which methods that can be used when immobilising enzymes 12/31/2022 by TDLV 162