ENZYME INHIBITION & FACTORS AFFECTING THE VELOCITY OF ENZYME ACTION

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ENZYME INHIBITION

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<ul><li> 1. Factors affecting enzyme activityGandham. Rajeev</li></ul><p> 2. Factors affecting enzyme activity The contact between the enzyme and substrate is the mostessential pre-requisite for enzyme activity1. Enzyme concentration2. Substrate concentration3. Temperature4. Hydrogen ion concentration (pH)5. Product concentration6. Presence of activators7. Time8. Light &amp; radiation 3. Enzyme concentration Enzyme Concentration: Rate of a reaction or velocity (V) is directly proportional to the enzymeconcentration, when sufficient substrate is present. Velocity of reaction is increased proportionately with the concentration ofenzyme, provided substrate concentration is unlimited 4. Substrate is a molecule on which enzyme acts. Velocity (Reaction rate) refers to change in the concentration ofsubstrate or reaction product (s) per unit time. It is expressed as moles/liter/sec. Maximum velocity (Vmax): It refers to maximum change in the product or substrate concentrationat a given enzyme concentration. 5. Vmax = Kcat (e) e-enzyme concentration &amp; Kcat is catalytic rate constant. Kcat (catalytic rate constant) defined as the number of substratesmolecules formed by each enzyme molecule in unit time. Expressed as moles produced/mol enzyme/time. 6. Effect of enzyme concentration 7. Effect of Substrate Concentration Increase in the substrate concentration gradually increases thevelocity of enzyme reaction within the limited range of substratelevels. A rectangular hyperbola is obtained when velocity is plotted againstthe substrate concentration Three distinct phases of the reaction are observed in the graph (A-linear;B-curve; C-almost unchanged. 8. Effect of Substrate Concentration 9. Explanation At lower concentrations of substrate (point A in the curve), some enzymemolecules are remaining idle. As substrate is increased, more and more enzyme molecules are working. At half-maximal velocity, 50% enzymes are attached with substrate (point Bin the curve). As more substrate is added, all enzyme molecules are saturated (point C). 10. Further increase in substrate cannot make any effect in the reactionvelocity (point D). The maximum velocity obtained is called Vmax. It represents the maximum reaction rate attainable in presence of excesssubstrate (at substrate saturation level). 11. Michaelis-Mention Equation Michaelis-Mention equation is a rate equation for reaction kinetics inenzyme catalysed reaction Written asV max (S)Km + SV = 12. Michaelis-mention Plot The velocity of enzyme catalysed reactions is altered as the substrateconcentration is increased. First order reaction: At low substrate concentration, velocity increases proportionally asthe concentration of the substrate is increased. 13. Mixed order reaction: When the concentration of the substrate is further increased (at midsubstrate concentration), the velocity increases, but notproportionally to substrate concentration. Zero order reaction: At high substrate concentration, the velocity is maximum &amp; isindependent of substrate concentration. 14. Enzyme kinetics &amp; Km value The enzyme (E) reacts with substrate (S) to form unstable enzyme-substrate(ES) complex. The ES complex is either converted to product (P) or can dissociateback to enzyme (E) &amp; substrate (S).Substrate (S) + Enzyme (E) Enzyme substrate (ES) Product (P) + Enzyme (E) 15. K1 K3E + S ES E + PK2 K1,K2 &amp; K3 are velocity constants. Km, Michaelis-mention constant is given by the formulaKm =K2 + K3K1 16. Michaelis-mention set up mathematical expressions for the rate of all the threereactions in the equation. V as the initial rate of reaction (velocity) S as the initial concentration of the substrate V max as the maximum velocity attained with high substrate concentration when allthe enzyme molecules are occupied. Km as Michaelis-mention constantV =V max (S)Km + (S) 17. Measured velocity (V) is equal to Vmax. So, V max =V max (S)Km + (S)Km + (S) =2V max (S)V maxKm + (S) = 2 (S)Km = (S)K stands for constant &amp; M stands for Michaelis 18. Michaelis constant The formation of enzyme - substrate complex is a reversible reaction, while thebreakdown of the complex to enzyme + product is irreversible. 50% velocity in Y axis is extrapolated to the corresponding point on X-axis, which givesthe numerical value of Km. The lesser the numerical value of Km, the affinity of the enzyme for the substrate ismore. E.g: Km of glucokinase is 10 mmol/L and hexokinase is 0.05 mmol/L. 50% molecules of hexokinase are saturated even at a lower concentration of glucose. Hexokinase has more affinity for glucose than glucokinase. 19. Effect of enzyme concentration on Km 20. Salient features of Km Km value is substrate concentration (expressed in moles/L) at half-maximal velocity. It denotes that 50% of enzyme molecules are bound with substrate molecules atthat particular substrate concentration. Km is independent of enzyme concentration. If enzyme concentration is doubled, the Vmax will be double. But the Km will remain exactly same. In other words, irrespective of enzyme concentration, 50% molecules are bound tosubstrate at that particular substrate concentration. 21. Km is the Signature of the Enzyme. Km value is thus a constant for an enzyme. It is the characteristic feature of a particular enzyme for a specificsubstrate. The affinity of an enzyme towards its substrate is inversely related tothe dissociation constant, Kd for the ES complex. Km denotes the affinity of enzyme for substrate. The lesser the numerical value of Km, the affinity of the enzyme for theenzyme for the substrate is more. 22. Double reciprocal plot Sometimes it is impractical to achieve high substrate concentrations to reachthe maximal velocity conditions. So, Vmax or Km may be difficult to determine. The experimental data at lower concentrations is plotted as reciprocals. The straight line thus obtained is extrapolated to get the reciprocal of Km. Called as LineweaverBurk Plot or Double Reciprocal Plot which can bederived from the Michaelis-Menten equation 23. Lineweaver-Burk plot 24. Effect of Temperature The velocity of enzyme reaction increases when temperature of the mediumis increased; reaches a maximum and then falls (Bell shaped curve). The temperature at which maximum amount of the substrate is converted tothe product per unit time is called the optimum temperature. Temperature is increased, more molecules get activation energy, ormolecules are at increased rate of motion. Their collision probabilities are increased and so the reaction velocity isenhanced. 25. Temperature coefficient Q10 The temperature coefficient (Q10) is the factor by which the rate ofcatalysis is increased by a rise in 10C. Generally, the rate of reaction of most enzymes will double by a rise in10C. When temperature is more than 50C, heat denaturation andconsequent loss of tertiary structure of protein occurs. Activity of the enzyme is decreased. 26. Most human enzymes have the optimum temperature around 37C. Certain bacteria living in hot springs will have enzymes with optimumtemperature near 100C. 27. Effect of Temperature 28. Effect of pH Each enzyme has an optimum pH (usually pH between 6 and 8). On both sides of which the velocity will be drastically reduced. The graph will show a bell shaped curve The pH decides the charge on the amino acid residues at the active site. The net charge on the enzyme protein would influence substrate binding andcatalytic activity. Optimum pH may vary depending on the temperature, concentration ofsubstrate, presence of ions etc. Pepsin (optimum pH 1-2); ALP (optimum pH 9-10) &amp; acid phosphatase (4-5) 29. Effect of pH 30. Effect of product concentration The accumulation of reaction products generally decreases the enzymevelocity. For certain enzymes, the products combine with the active site of enzymeand form a loose complex and, thus, inhibit the enzyme activity. In the living system, this type of inhibition is generally prevented by a quickremoval of products formed 31. Effect of activators Some of the enzymes require certain inorganic metallic cations like Mg2+,Mn2+, Zn2+, Ca2+, Co2+, Cu2+, Na+, K+, for their optimum activity Anions are also needed for enzyme activity e.g. chloride ion for amylase Metals function as activators of enzyme velocity through various mechanismscombining with the substrate, formation of ES-metal complex, directparticipation in the reaction and bringing a conformational change in theenzyme. 32. Two categories of enzymes requiring metals for their activity Metal-activated enzymes Metalloenzyme Metal-activated enzymes: The metal is not tightly held by the enzyme and can be exchangedeasily with other ions. e.g. ATPase (Mg2+ and Ca2+) &amp; Enolase (Mg2+) 33. Metalloenzyme: These enzymes hold the metals rather tightly which are not readilyexchanged. e.g. Alcohol dehydrogenase, carbonic anhydrase, alkaline phosphatase,carboxypeptidase and aldolase contain zinc. Phenol oxidase (copper) Pyruvate oxidase (manganese) Xanthine oxidase (molybdenum) Cytochrome oxidase (iron and copper) 34. Effect of time Under ideal and optimal conditions (like pH, temperature etc.), the timerequired for an enzyme reaction is less. Variations in the time of the reaction are generally related to thealterations in pH and temperature. 35. Effect of light and radiation Exposure of enzymes to ultraviolet, beta, gamma &amp; X-raysinactivates certain enzymes due to the formation of peroxides.e.g. UV rays inhibit salivary amylase activity 36. Enzyme inhibition 37. Enzyme inhibitor Enzyme inhibitor is defined as a substance, which binds with the enzymeand brings about a decrease in catalytic activity of that enzyme. They 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 38. Type of Enzyme InhibitorsReversibleIrreversibleType ofInhibitorsCompetitiveUncompetitiveNon- CompetitiveActive SiteDirectedSuicide / kcatInhibitors 39. Reversible inhibition The inhibitor binds non-covalently with enzyme and the enzyme inhibitioncan be reversed if the inhibitor is removed. Binding is weak and thus, inhibition is reversible. Do not cause any permanent changes in the enzyme Subtypes: Competitive &amp; Non-competitive Inhibition 40. Competitive inhibition The inhibitor (I) molecules resembles the real substrate (S) Also called as substrate analogue inhibition Binds to active site forms EI complex. EI complex cannot rive rise to product formation. As long as the competitive inhibitor holds the active site, the enzyme is not available forthe substrate to bind. Relative concentrations of S, I determine inhibition.EESEIE + PNo product formation 41. Binding of S &amp; I in different Situations Classical Competitive Inhibition (S &amp; I compete for the same bindingsite)Enzyme 42. Binding of I to a distinct inhibitor site causes a conformational change inthe enzyme that distorts or masks the S binding site or vice versa.I SEnzymeIEnzymeSIEnzymeS 43. A competitive inhibitor diminishes the rate of catalysis by reducing theproportion of enzyme molecules bound to a substrate. Competitive inhibition can be relieved by increasing the substrateconcentration &amp; maximum velocity is regained. A higher substrate concentration is therefore needed to achieve ahalfmaximum rate, Km increases High concentrations of the substrate displace the inhibitor again. The V max, not influenced by this type of inhibition. 44. E. g. Malonate structural analog of succinate-inhibits succinatedehydrogenase. 45. The effect of enzyme inhibitionSuccinateFumarate + 2H++ 2e-Succinate dehydrogenaseCH2COOHCHCOOHCOOHCH2COOH CHCOOHCOOHCH2Malonate 46. The compounds malonic acid, glutaric acid and oxalic acid, have structuralsimilarity with succinic acid and compete with the substrate for binding atthe active site of SDH. Antimetabolites: These chemical compounds that block the metabolic reactions by theirinhibitory action on enzymes. Antimetabolites are usually structural analogues of substrates and thus arecompetitive inhibitors. They are in use for cancer therapy, gout etc. 47. Examples of competitive inhibitionEnzyme Substrate Competitive inhibitorSuccinate Dehydrogenase Succinate MalonateDihydrofolate Reductase 7,8-dihydrofolate AminopterinXanthine Oxidase Hypoxanthine AllopurinolAcetyl cholinesterase Acetylcholine SuccinylcholineLactate Dehydrogenase Lactate OxamateHMG CoA Reductase HMG Co A HMG 48. Reversible, Competitive Inhibitors 49. In the presence of a competitive inhibitor Km increasesV max unchangedNo inhibitor+ C InhibitorVmax VmaxKm Kmapp[s]v 50. Lineweaver Burk plot[I]2[I]11Kmapp1Km In the presence of acompetitive inhibitor Kmincreases V max unchanged 51. Non-Competitive Inhibition The inhibitor binds at a site other than the active site on the enzyme &amp;causes conformational changes on enzymes or some times it may reactwith functional group at the active site &amp; inactivates the enzyme. This binding impairs the enzyme function. Inhibitor has no structural resemblance with the substrate. There is no competition for the active site of the enzyme molecule. 52. There usually exists a strong affinity for the inhibitor to bind at thesecond site. The inhibitor does not interfere with the enzyme-substrate binding. But the catalysis is prevented, possibly due to a distortion in the enzymeconformation The inhibitor generally binds with the enzyme as well as the ES complex. Km value is unchanged &amp; V max is lowered. 53. Heavy metal ions (Ag+, Pb2+, Hg2+ etc.) can non-competitively inhibit the enzymes bybinding with cysteinyl sulfhydryl groups &amp; inactivates the enzymes. Heavy metals also form covalent bonds with carboxyl groups &amp; histidine, results inirreversible inhibition. Non-competitive inhibition is also called as enzyme poisonsE + S ES+IEI + SE + P+IEIS 54. Non-Competitive InhibitionEnzyme EnzymeEnzyme EnzymeSISI 55. Non-Competitive InhibitionNo inhibitor+ NC InhibitorVmaxVmax i VmaxKm [s]v Vmax iVmax = Decreases.Km = Unchanged 56. Lineweaver Burk Plot[I]2[I]1No Inhibitor1Vmax1Vmaxi1Km1/[s]1/v Km value is unchanged V max is lowered 57. Comparison between competitive &amp; Non-competitive inhibitionCompetitive Inhibition Non-competitive InhibitionActing on Active site May or may notStructure of inhibitor Substrate analogue Unrelated moleculeInhibition is Reversible Generally IrreversibleExcess Substrate Inhibition Relieved No effectKm Increased No ChangeV max No Change DecreasedSignificance Drug Action Toxicological 58. Uncompetitive Inhibition Here inhibitor does not have any affinity for the active site of enzyme. Inhibitor binds only with enzyme-substrate complex; but not with freeenzyme. Both V max and Km are decreased 59. UC Inhibition is rare in single-substrate reactions. E.g. Phenylalanine inhibits alkaline phosphatase in intestinal cells...</p>