ENZYME INHIBITION & FACTORS AFFECTING THE VELOCITY OF ENZYME ACTION

ENZYME INHIBITION

1. Factors affecting enzyme activity Gandham . Rajeev

2. Factors affecting enzyme activity The contact between the enzyme and substrate is the most essential pre-requisite for enzyme activity Enzyme concentration Substrate concentration Temperature Hydrogen ion concentration ( pH) Product concentration Presence of activators Time Light & radiation

3. Enzyme concentration Enzyme Concentration: Rate of a reaction or velocity (V) is directly proportional to the enzyme concentration, when sufficient substrate is present. Velocity of reaction is increased proportionately with the concentration of enzyme, provided substrate concentration is unlimited

4. Substrate is a molecule on which enzyme acts. Velocity (Reaction rate) refers to change in the concentration of substrate 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 concentration at a given enzyme concentration.

5. Vmax = Kcat (e) e-enzyme concentration & Kcat is catalytic rate constant. Kcat (catalytic rate constant) – defined as the number of substrates molecules 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 the velocity of enzyme reaction within the limited range of substrate levels. A rectangular hyperbola is obtained when velocity is plotted against the 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 enzyme molecules 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 B in 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 reaction velocity (point D). The maximum velocity obtained is called Vmax . It represents the maximum reaction rate attainable in presence of excess substrate (at substrate saturation level).

11. Michaelis -Mention Equation Michaelis -Mention equation is a rate equation for reaction kinetics in enzyme catalysed reaction Written as V max (S) Km + S V =

12. Michaelis -mention Plot The velocity of enzyme catalysed reactions is altered as the substrate concentration is increased. First order reaction: At low substrate concentration, velocity increases proportionally as the concentration of the substrate is increased.

13. Mixed order reaction: When the concentration of the substrate is further increased (at mid substrate concentration), the velocity increases, but not proportionally to substrate concentration. Zero order reaction: At high substrate concentration, the velocity is maximum & is independent of substrate concentration.

14. Enzyme kinetics & 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 dissociate back to enzyme (E) & substrate (S). Substrate (S) + Enzyme (E) Enzyme substrate (ES) Product (P) + Enzyme (E)

15. K1,K2 & K3 are velocity constants. Km, M ichaelis -mention constant is given by the formula… E + S ES E + P K2 K1 K3 Km = K2 + K3 K1

16. Michaelis -mention set up mathematical expressions for the rate of all the three reactions 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 all the enzyme molecules are occupied. Km as M ichaelis -mention constant V = V max (S) Km + (S)

17. Measured velocity (V) is equal to ½ Vmax . So, ½ V max = V max (S) Km + (S) Km + (S) = 2V max (S) V max Km + (S) = 2 (S) Km = (S) K stands for constant & M stands for Michaelis

18. Michaelis constant The formation of enzyme - substrate complex is a reversible reaction , while the breakdown of the complex to enzyme + product is irreversible . 50% velocity in Y axis is extrapolated to the corresponding point on X-axis , which gives the numerical value of Km. The lesser the numerical value of Km, the affinity of the enzyme for the substrate is more. 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. H exokinase 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 at that 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 to substrate 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 specific substrate. The affinity of an enzyme towards its substrate is inversely related to the 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 the substrate is more.

22. Double reciprocal plot Sometimes it is impractical to achieve high substrate concentrations to reach the maximal velocity conditions. So , ½Vmax 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 Lineweaver –Burk Plot or Double Reciprocal Plot which can be derived from the Michaelis-Menten equation

23. Lineweaver -Burk plot

24. Effect of Temperature The velocity of enzyme reaction increases when temperature of the medium is increased; reaches a maximum and then falls (Bell shaped curve). The temperature at which maximum amount of the substrate is converted to the product per unit time is called the optimum temperature. Temperature is increased, more molecules get activation energy, or molecules are at increased rate of motion. Their collision probabilities are increased and so the reaction velocity is enhanced .

25. T emperature coefficient Q10 The temperature coefficient (Q10) is the factor by which the rate of catalysis is increased by a rise in 10°C. Generally, the rate of reaction of most enzymes will double by a rise in 10°C. W hen temperature is more than 50°C, heat denaturation and consequent loss of tertiary structure of protein occurs. Activity of the enzyme is decreased.

26. Most human enzymes have the optimum temperature around 37°C. Certain bacteria living in hot springs will have enzymes with optimum temperature near 100°C .

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 and catalytic activity . Optimum pH may vary depending on the temperature, concentration of substrate , presence of ions etc. Pepsin (optimum pH 1-2 ); ALP (optimum pH 9-10) & acid phosphatase (4-5 )

29. Effect of pH

30. Effect of product concentration The accumulation of reaction products generally decreases the enzyme velocity . For certain enzymes, the products combine with the active site of enzyme and form a loose complex and, thus, inhibit the enzyme activity. In the living system, this type of inhibition is generally prevented by a quick removal of products formed

31. Effect of activators Some of the enzymes require certain inorganic metallic cations like Mg 2 + , Mn 2+ , Zn 2 + , Ca 2 + , Co 2+ , Cu 2 + , 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 mechanisms combining with the substrate, formation of ES-metal complex, direct participation in the reaction and bringing a conformational change in the enzyme .

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 exchanged easily with other ions. e.g. ATPase (Mg 2+ and Ca 2+ ) & Enolase (Mg 2+ )

33. Metalloenzyme : These enzymes hold the metals rather tightly which are not readily exchanged . 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 time required for an enzyme reaction is less. Variations in the time of the reaction are generally related to the alterations in pH and temperature.

35. Effect of light and radiation Exposure of enzymes to ultraviolet, beta, gamma & X-rays inactivates 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 enzyme and 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 Inhibitors Reversible Irreversible Type of Inhibitors Competitive Uncompetitive Non- Competitive Active Site Directed Suicide / k cat Inhibitors

39. Reversible inhibition The inhibitor binds non-covalently with enzyme and the enzyme inhibition can 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 & 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 for the substrate to bind. Relative concentrations of S, I determine inhibition. E ES E I E + P +S + I No product formation

41. Binding of S & I in different Situations Classical Competitive Inhibition (S & I compete for the same binding site) S I Enzyme

42. Binding of I to a distinct inhibitor site causes a conformational change in the enzyme that distorts or masks the S binding site or vice versa. Enzyme I S Enzyme I S Enzyme I S

43. A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate. Competitive inhibition can be relieved by increasing the substrate concentration & maximum velocity is regained. A higher substrate concentration is therefore needed to achieve a halfmaximum 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 succinate dehydrogenase.

45. The effect of enzyme inhibition Succinate Fumarate + 2H + + 2e - Succinate dehydrogenase CH 2 COOH CH 2 COOH CHCOOH CHCOOH COOH COOH CH 2 Malonate

46. The compounds malonic acid, glutaric acid and oxalic acid , have structural similarity with succinic acid and compete with the substrate for binding at the active site of SDH. Antimetabolites: These chemical compounds that block the metabolic reactions by their inhibitory action on enzymes. Antimetabolites are usually structural analogues of substrates and thus are competitive inhibitors. They are in use for cancer therapy , gout etc.

47. Examples of competitive inhibition Enzyme Substrate Competitive inhibitor Succinate Dehydrogenase Succinate Malonate Dihydrofolate Reductase 7,8-dihydrofolate Aminopterin Xanthine Oxidase Hypoxanthine Allopurinol Acetyl cholinesterase Acetylcholine Succinylcholine Lactate Dehydrogenase Lactate Oxamate HMG CoA Reductase HMG Co A HMG

48. Reversible, Competitive Inhibitors

49. In the presence of a competitive inhibitor K m increases V max unchanged No inhibitor + C Inhibitor V max ½ V max K m Km app [s] v

50. Lineweaver Burk plot [I] 2 [I] 1 1 Km app 1 Km Slope = Km Vmax ( 1+ ) [I] Ki In the presence of a competitive inhibitor K m increases V max unchanged

51. Non-Competitive Inhibition The inhibitor binds at a site other than the active site on the enzyme & causes conformational changes on enzymes or some times it may react with functional group at the active site & 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 the second site. The inhibitor does not interfere with the enzyme-substrate binding . But the catalysis is prevented, possibly due to a distortion in the enzyme conformation The inhibitor generally binds with the enzyme as well as the ES complex. Km value is unchanged & V max is lowered.

53. Heavy metal ions (Ag + , Pb 2+ , Hg 2+ etc.) can non-competitively inhibit the enzymes by binding with cysteinyl sulfhydryl groups & inactivates the enzymes. Heavy metals also form covalent bonds with carboxyl groups & histidine , results in irreversible inhibition. Non-competitive inhibition is also called as enzyme poisons E + S ES + I E I + S E + P + I E I S

54. Non-Competitive Inhibition Enzyme Enzyme Enzyme Enzyme S I S I

55. Non-Competitive Inhibition No inhibitor + NC Inhibitor V max ½ V max K m [s]  v ½ V max i V max i Vmax = Decreases. Km = Unchanged

56. Lineweaver – Burk Plot [I] 2 [I] 1 No Inhibitor 1 V max 1 V maxi 1 Km 1/[s]  1/v Km value is unchanged V max is lowered

57. Comparison between competitive & Non-competitive inhibition Competitive Inhibition Non-competitive Inhibition Acting on Active site May or may not Structure of inhibitor Substrate analogue Unrelated molecule Inhibition is Reversible Generally Irreversible Excess Substrate Inhibition Relieved No effect Km Increased No Change V max No Change Decreased Significance 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 free enzyme. 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 It is common in multi-substrate reactions E + S E S E + P + I ESI

60. Uncompetitive Inhibition Enzyme Enzyme S Enzyme I S

61. Uncompetitive Inhibition No inhibitor + UC Inhibitor V max ½ V max K m [s]  ½ V max i V max i Vmax = Decreases Km = Decreases Km app v

62. In this type, Inhibitor binds at or near the active site of the enzyme irreversibly, usually by covalent bonds, so that it can’t subsequently dissociate from the enzyme The I destroys as essential functional group on the enzyme that participates in normal S binding or catalytic action. As a result the enzyme is permanently inactive Compounds which irreversibly denature the enzyme protein or cause non-specific inactivation of the active site are not usually regarded as irreversible inhibitors. Irreversible inhibition

63. These inhibitors are toxic poisonous substances. Iodoacetate : It is an irreversible inhibitor of the enzymes like papain and glyceraldehyde 3-phosphate dehydrogenase Iodoacetate combines with sulfhydryl (-SH) groups at the active site of these enzymes and makes them inactive. Diisopropyl fluorophosphafe (DFP) is a nerve gas developed by the Germans during Second World War. DFP irreversibly binds with enzymes containing serine at the active site, e.g. serine proteases, acetylcholine esterase. Examples

64. Examples DFP ( Diisopropylphosphofluoridate ) is a nerve poison. It inactivates acetylcholinesterase that plays an important role in the transmission of nerve impulses. E CH 2 -OH + F—P=O E CH 2 -O- F—P=O + HF OCH(CH 3 ) 2 OCH(CH 3 ) 2 OCH(CH 3 ) 2 OCH(CH 3 ) 2 DFP Catalytically inactive enzyme

65. Disulfiram ( Antabuse )s a drug used in the treatment of alcoholism . lt irreversibly inhibits the enzyme aldehyde dehydrogenase. Alcohol addicts , when treated with disulfiram become sick due to the accumulation of acetaldehyde , leading to alcohol avoidance

66. Suicidal inhibition This is a special type of irreversible inhibition. Also called as mechanism based inactivation. In this case , the original inhibitor (the structural analogue/competitive inhibitor ) is converted to a more effective inhibitor with the help of same enzyme that ought to be inhibited. The formed inhibitor binds irreversibly with the enzyme. Allopurinol, an inhibitor of xanthine oxidase , gets converted to alloxanthine , a more effective inhibitor of this enzyme .

67. A suicide inhibitor is a relatively inert molecule that is transformed by an enzyme at its active site into a reactive compound that irreversibly inactivates the enzyme They are substrate analogs designed so that via normal catalytic action of the enzyme, a very reactive group is generated. The latter forms a covalent bond with a nearby functional group within the active site of the enzyme causing irreversible inhibition. Such inhibitors are called suicide inhibitors because the enzyme appears to commit suicide. e.g . FdUMP is a suicide inhibitor of thymidylate synthase. Suicidal inhibition

68. The use of certain purine and pyrimidine analogues in cancer therapy is also explained on the basis suicide inhibition. 5-fluorouracil gets converted to fluorodeoxyuridylate which inhibits the enzyme thymidylate synthase, and thus nucleotides synthesis

69. During thymidylate synthesis, N 5 ,N 10 - methyleneTHF is converted to 7,8-dihydrofolate; methyleneTHF is regenerated in two steps

70. Allosteric regulation The catalytic activity of certain regulatory enzymes is modified by certain low molecular weight substances or molecules known as allosteric effectors. Allosteric enzyme has one catalytic site where the substrate binds and another separate allosteric site where the modifier binds ( allo = other ) Allosteric and substrate binding sites may or may not be physically adjacent. The binding of the regulatory molecule can either enhance the activity of the enzyme ( allosteric activation), or inhibit the activity of the enzyme (allosteric inhibition ).

71. The binding of substrate to one of the subunits of the enzyme may enhance substrate binding by other subunits. This effect is said to be positive co- operativity If the binding of substrate to one of the subunits decreases the activity of substrate binding by other sites, the effect is called negative co- operativity . In most cases , a combination is observed , resulting in a sigmoid shaped curve

72. The switch: Allosteric inhibition Allosteric means “other site” E Active site Allosteric site

73. Switching off These enzymes have two receptor sites One site fits the substrate like other enzymes The other site fits an inhibitor molecule Inhibitor fits into allosteric site Substrate cannot fit into the active site Inhibitor molecule

74. Allosteric inhibition

75. Salient Features, Allosteric Inhibition The inhibitor is not a substrate analogue It is partially reversible, when excess substrate is added . Km is usually increased & V max is reduced . The effect of allosteric modifier is maximum at or near substrate concentration equivalent to Km.

76. When an inhibitor binds to the allosteric site, the configuration of catalytic site is modified such that substrate cannot bind properly. Most allosteric enzymes possess quaternary structure. They are made up of subunits, e.g. Aspartate transcarbamoylase has 6 subunits and pyruvate kinase has 4 subunits

77. A llosteric enzymes Enzyme Allosteric Inhibitor Allosteric Activator ALA synthase Heme Aspartate transcarbamoylase CTP ATP HMGCoA-reductase Cholesterol Phosphofructokinase ATP, citrate AMP, F-2,6-P Pyruvate carboxylase ADP AcetylCoA Acetyl CoA carboxylase AcylCoA Citrate Citrate synthase ATP Carbamoyl phosphate synthetase I NAG Carbamoyl phosphate synthetase II UTP

78. For understanding the regulation of enzyme activity within the living cells To elucidate the kinetic mechanism of an enzyme catalyzing a multi-substrate reaction Useful in elucidating the cellular metabolic pathways by causing accumulation of intermediates Identification of the catalytic groups at the active site Provide information about substrate specificity of the enzyme Importance of Enzyme Inhibition

79. Regulation of enzyme activity Allosteric regulation Activation of latent enzymes Compartmentation of metabolic pathways Control of enzyme synthesis Enzyme degradation lsoenzymes

80. Allosteric Regulation or Allosteric Inhibition Enzymes possess additional sites, known as allosteric sites besides the active site. Such enzymes are known as allosteric enzymes. The allosteric sites are unique places on the enzyme molecule Allosteric effectors: The catalytic activity of certain regulatory enzymes is modified by certain low molecular weight substances or molecules known as allosteric effectors or modifiers bind at the allosteric site and regulate the enzyme activity.

81. The allosteric effectors may be positive or negative effectors The enzyme activity is increased when a positive (+) allosteric effector binds at the allosteric site known as activator site . A negative (-) allosteric effector binds at the allosteric site called inhibitor site and inhibits the enzyme activity. Classes of allosteric enzyme: They are divided into two classes based on the influence of allosteric effector on Km and V max

82. K-class of allosteric enzymes: The allosteric inhibitor increases the Km and not the V max. Double reciprocal plots, similar to competitive inhibition are obtained e.g. phosphofructokinase. V-class of allosteric enzymes: The allosteric inhibitor decreases the V max and not the Km. Double reciprocal plots resemble that of non-competitive inhibition e.g. acetyl CoA carboxylase

83. Feedback regulation The process of inhibiting the first step by the final product, in a series of enzyme catalysed reactions of a metabolic pathway is referred to as feedback regulation. The very first step (A to B) by the enzyme is the most effective for regulating the pathway, by the final end product D. This type of control is often called negative feedback regulation A B C D

84. Feedback regulation Carbamoyl phosphate + Aspartate Carbamoyl Aspartate + Pi Cytidine triphosphate (CTP) Aspartate transcarbamylase Feedback control

85. Activation of latent enzymes Some enzymes are synthesized as Proenzymes or zymogens which undergo irreversible covalent activation by the breakdown of one or more peptide bonds Chymotrypsinogen pepsinogen and plasminogen, are respectively- converted to the active enzymes chymotrypsin, pepsin and plasmin. Certain enzymes exist in the active and inactive forms which are interconvertible The inter-conversion is brought about by the reversible covalent modifications, namely phosphorylation and dephosphorylation , and oxidation and reduction of disulfide bonds

86. Examples There are some enzymes which are active in dephosphorylated state and become inactive when phosphorylated e.g. glycogen synthase , acetyl CoA carboxylase . A few enzymes are active only with sulfhydryl (- SH) groups E.g . succinate dehydrogenase , urease. Glutathione bring about the stability of these enzymes.

87. Compartmentation G enerally , the synthetic (anabolic) and b reakdown (catabolic ) pathways are operative in different cellular organelle. E.g. Enzymes for fatty acid synthesis are found in the cytosol whereas enzymes for fatty acid oxidation are present in the mitochondria

88. Control of enzyme synthesis Most of the enzymes, the rate limiting ones, are present in very low concentration. Many rate limiting enzymes have short half-lives This helps in the efficient regulation of the enzyme levels. Constitutive enzymes (house-keeping enzymes )- T he levels of which are not controlled and remain fairly constant. Adaptive enzymes - Their concentrations increase or decrease as per body needs and are well-regulated. The synthesis of enzymes ( proteins) is regulated by the genes.

89. Induction and repression Induction is used to represent increased synthesis of enzyme while repression indicates its decreased synthesis . Induction or repression which ultimately determines the enzyme concentration at the gene level through the mediation of hormones or other substance. E.g of Induction: The hormone insulin induces the synthesis of glycogen synthetase , glucokinase , phosphofructokinase and pyruvate kinase . All these enzymes are involved in the utilization of glucose. The hormone cortisol induces the synthesis of many enzymes e.g. pyruvate carboxylase, tryptophan oxygenase and tyrosine aminotransferase

90. Examples of repression: In many instances , substrate can repress the synthesis of enzyme. Pyruvate carboxylase is a key enzyme in the synthesis of glucose from non-carbohydrate sources like pyruvate and amino acids . lf there is sufficient glucose available, there is no necessity for its synthesis. This is achieved through repression of pyruvate carboxylase b y glucose.

91. Enzyme degradation Every enzyme has half-life. It is in days while for others in hours or in minutes , e.g . LDH4 - 5 to 6 days ; LDH1 - 8 to 12 hours; Amylase -3 to 5 hours The key and regulatory enzymes are most rapidly degraded . lf not needed, they immediately disappear and, when required, they are quickly synthesized

92. Units of enzyme activity Katal : One kat denotes the conversion of one mole substrate per second ( mol /sec). Activity may also be expressed as millikatals ( mkat ), microkatals (µ kat ) International Units ( lU ): One Sl unit or International Unit ( lU ) is defined as the amount of enzyme activity that catalyses the conversion of one micromol of substrate per minute. Sl units and katal are interconvertible

93. Non-protein enzymes Ribozymes are a group of ribonucleic acids that function as biological catalysts, and they are regarded as non-protein enzymes . RNA molecules are known to adapt a tertiary structure just as in the case of proteins The specific conformation of RNA may be responsible for its function as biocatalyst.

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