Enzymes

ENZYMES

1. ENZYMES

2. Enzymes may be defined as biocatalysts synthesized by living cells. They are protein in nature, colloidal and thermolabile in character, and specific in action. A catalyst is defined as a substance that increases the velocity or rate of a chemical reaction without itself undergoing any change in the overall process. Friedrich Wilhelm Kuhne (1878) – given the name ENZYME (En = in ; Zyme = yeast) James B. Sumner (1926 ) - first isolated & crystallized urease from jack bean and identified protein .

3. NOMENCLATURE AND CLASSIFICATION 1. Substrate acted upon by the enzyme: named the enzymes by adding the “ ase ” in the name of the substrate catalyzed Eg., carbohydrates – carbohydrases proteins - proteinases , lipids – lipases nucleic acids - nucleases A few of the names were even more specific like Eg., maltase - acting upon maltose sucrase - upon sucrose urease - upon urea etc .

4. 2. Type of reaction catalyzed: The enzymes are named by adding the “ ase ” in the name of the reaction Eg., Hydrolases - catalyzing hydrolysis Isomerases – isomerization Oxidases – catalyzing oxidation Dehydrogenases – catalyzing dehydrogenation Transaminases – catalyzing transamination Phosphorylases - catalyzing phosphorylation etc.

5. 3. Substrate acted upon and type of reaction catalyzed: The names of some enzymes give clue of both the substrate utilized and the type of reaction catalyzed. Eg., succinic dehydrogenase – catalyzes dehydrogenation of the substrate succinic acid. L-glutamic dehydrogenase – catalyzes dehydrogenation reaction involving L-glutamic acid.

6. 4. Substance that is synthesized : A few enzymes have been named by adding the “ ase ” to the name of the substance synthesized Eg., rhodonase - forms rhodonate from hydrocyanic acid and sodium thiosulphate fumarase - forms fumarate from L-malate.

7. 5. Endoenyzmes and exoenyzmes: enzymes that act within the cells in which they are produced - intracellular enzymes or endoenzymes Eg., plant enzymes and metabolic enzymes Enzymes which are liberated by living cells, catalyze useful reactions outside the cell in its environment - extracellular enzymes or exoenzymes Eg., bacterial enzymes, fungal enzymes, digestive tract enzymes

8. INTERNATIONAL UNION OF BIOCHEMISTRY (IUB) NOMENCLAURE AND CLASSIFICATION The chemical reaction catalyzed is the specific property which distinguishes one enzyme from another. In 1961, IUB used this criterion as a basis for the classification and naming of enzymes . According to IUB, the reactions and the enzymes catalyzing them are divided into 6 major classes, each with 4 to 13 subclasses.

9. Oxidoreductases Transferases Hydrolases Lyases or Desmolases Isomerases Ligases or Synthetases

10. 1. Oxidoreductases: Enzymes which bring about oxidation-reduction reactions between two substrates. Groups to be present in the substrate: CH—OH , C=O, CH—CH, CH—NH 2 and CH=NH groups Eg., Alcohol dehydrogenase, Acyl-CoA dehydrogenase, Cytochrome oxidase etc.,

11. 2. Transferases: Enzymes which catalyze the transfer of a group , G (other than hydrogen) between a pair of substrates, S and S′ are called transferases S—G + S′ -----------→ S + S′— G These enzymes catalyze the transfer of one-carbon groups, aldehydic or ketonic residues and acyl, glycosyl , alkyl, phosphorus or sulfur-containing groups Eg., Acyltransferases, Glycosyltransferases, Hexokinase

12. 3. Hydrolases: These catalyze the hydrolysis of their substrates by adding constituents of water across the bond they split. The substrates include ester, glycosyl, ether, peptide, acid-anhydride , C—C , halide and P—N bonds . e.g., glucose-6-phosphatase, pepsin, trypsin, esterases , glycoside hydrolases

13. 4. Lyases (Desmolases): These are those enzymes which catalyze the removal of groups from substrates by mechanisms other than hydrolysis, leaving double bonds . These include enzymes acting on C—C, C—O, C—N, C—S and C—halide bonds . Eg., Aldolase, Fumarase, Histidase etc.,

14. 5. Isomerases: These catalyze interconversion of optical, geometric or positional isomers by intramolecular rearrangement of atoms or groups . Eg., Alanine racemase , Retinene isomerase , Glucosephosphate isomerase etc.,

15. 6. Ligases: These are the enzymes catalyzing the linking together of two compounds utilizing the energy made available due to simultaneous breaking of a pyrophosphate bond in ATP or a similar compound. This category includes enzymes catalyzing reactions forming C—O, C—S, C—N and C—C bonds . Eg., Acetyl-CoA synthetase , Glutamine synthetase etc.,

16. CHEMICAL NATURE OF ENZYMES Simple-protein enzymes. These contain simple proteins only e.g., urease, amylase, papain etc . 2 . Complex-protein enzymes. These contain conjugated proteins i.e., they have a protein part called apoenzyme and a nonprotein part called prosthetic group associated with the protein unit. The two parts constitute what is called a holoenzyme

17. The activity of an enzyme depends on the prosthetic group that is tightly associated with the apoenzyme . But sometimes the prosthetic group is loosely bound to the protein unit and can be separated by dialysis and yet indispensable for the enzyme activity . In that case, this dialyzable prosthetic group is called as a coenzyme (organic nature) or cofactor (inorganic nature).

18. COENZYMES The non-protein, organic, Iow molecular weight and dialysable substance associated with enzyme function is known as coenzyme . Coenzymes are often regarded as the second substrates or co-substrates , since they have affinity with the enzyme comparable with that of the substrate Types of coenzymes: B-complex vitamin coenzymes and non B-complex vitamin coenzymes

19. COFACTORS The non-protein, inorganic , Iow molecular weight and dialysable substance associated with enzyme function is known as cofactors. Most of the cofactors are metal ions Metal activated enzymes: In these enzymes, the metals form a loose and easily dissociable complex. Eg ., ATPase ( Mg 2+ and Ca 2 + , Enolase ( Mg 2 + )

20. Metalloenzymes: In this case metal ion is bound tightly to the enzyme and is not dissociated Eg., 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).

21. ACTIVE SITE The active site (or active center) of an enzyme represents as the small region at which the substrate binds and participates in the catalysis Salient features: The existence of active site is due to the tertiary structure of protein. Made up of amino acids which are far from each other in the linear sequence of amino acids.

22. Active sites are regarded as clefts or crevices or pockets occupying a small region in a big enzyme molecule . The active site is not rigid, it is flexible to promote the specific substrate binding Enzymes are specific in their function due to the existence of active sites.

23. Active site possesses a substrate binding site and a catalytic site. The coenzymes or cofactors on which some enzymes depend are present as a part of the catalytic site. The substrate binds at the active site by weak noncovalent bonds.

24. The commonly found amino acids at the active sites are serine(mostly found) , aspartate, histidine , cysteine, lysine, arginine, glutamate, tyrosine . The substrate binds the enzyme (E) at the active site to form enzyme-substrate complex ( ES ). The product (P) is released after the catalysis and the enzyme is available for reuse .

25. MODE OF ENZYME ACTION Two theories have been put forth to explain mechanism of enzyme-substrate complex formation 1. Lock and key model/ Fischer’s template Theory 2. Induced fit theory/ Koshland’s model

26. Lock and key model/ Fischer’s template Theory: Proposed by a Emil Fischer. Very first model proposed to explain an enzyme catalyzed reaction According to this model, the structure or conformation of the enzyme is rigid . The substrate fits to the binding site just as a key fits into the proper lock or a hand into the proper glove. Thus the active site of an enzyme is a rigid and pre-shaped template where only a Specific substrate can bind.

27. This model was not accepted because Does not give any scope for the flexible nature of enzymes Totally fails to explain many facts of enzymatic reactions Does not explain the effect of allosteric modulator

28. 2. Induced fit theory/ Koshland’s model: Koshland proposed this model The active site is not rigid and pre-shaped The interaction of the substrate with the enzyme induces a fit or a conformation changei n the enzyme , resulting in the formation of a strong substrate binding site. Further more the appropriate amino acids of the enzyme are repositioned to form the active site and bring about the catalysis

29. This model was accepted because: Has sufficient experimental evidence from the X-ray diffraction studies . This model also explains the action of allosteric modulators and competitive inhibition on enzymes

30. FACTORS AFFECTING ENZYME ACTION Concentration of the enzyme: As the concentration of the enzyme is increased , the velocity of the reaction proportionately increases. This property of enzyme is made use in determining the serum enzymes for the diagnosis of diseases .

31. Concentration of the Substrate : 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 ).

32. Order of reaction : When the velocity of the reaction is almost proportional to the substrate concentration, the rate of the reaction is said to be first order with respect to substrate. When the substrate concentration is much greater than Concentration of enzyme, the rate of reaction is independent of substrate concentration , and the reaction is said to be zero order.

33. Effect of temperature: Velocity of an enzyme reaction increases with increase in temperature up to a maximum and then declines. A bell-shaped curve is usually observed.

34. Temperature coefficient or Q10 is defined as increase in enzyme velocity when the temperature is increased by 10 o C . For a majority of enzymes, Q10 is 2 between 0"C and 40 o C . optimum temperature - 40 o C-45 o C . (However , a few enzymes e.g . venom phosphokinases, muscle adenylatek inase are active even at 100 o C . Some plant enzymes like urease have optimum activity around 60 o C.)

35. when the enzymes are exposed to a temperature above 50 o C , denaturation leading to derangement in the native (tertiary ) structure of the protein and active site are seen. Majority of the enzymes become inactive at higher temperature (above 70 o C ).

36. Effect of pH: Each enzyme has an optimum pH at which the velocity is maximum. Below and above this pH, the enzyme activity is much lower and at extreme pH, the enzyme becomes totally inactive

37. Most of the enzymes of higher organisms show optimum activity around neutral pH (6-8). There are , however , many exceptions like pepsin ( 1-2), acid phosphatase (4-5) and alkaline phosphatase(10-11). Enzymes from fungi and plants are most active in acidic pH (4-6 ). Hydrogen ions influence the enzyme activity by altering the ionic charges on the amino acids (particularly at the active site) and substrate.

38. Effect of product concentration ln the living system, this type of inhibition is generally prevented by a quick removal of products formed

39. 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 . Effect of light and radiation: Exposure of enzymes to ultraviolet, beta , gamma and X-rays inactivates certain enzymes. The inactivation is due to the formation of peroxides. e.g . UV rays inhibit salivary amylase activity.

40. ENZYME KINETICS/ MICHAELIS-MENTEN HYPOTHESIS Leonor Michaelis and Maud L. Menten (1913), while studying the hydrolysis of sucrose catalyzed by the enzyme invertase , proposed this theory . According to this theory From the above equation theoretically one can explain the kinetics of the enzyme reaction, but practically not For this reason Micheali and Menten proposed an equation.

41. From that equation, these immeasurable quantities were replaced by those which could be easily measured experimentally. Following symbols may be used for deriving Michaelis -Menten equation : ( E t ) = total concentration of enzyme ( S) = total concentration of substrate ( ES) = concentration of enzyme-substrate complex ( E t ) − (ES) = concentration of free enzyme

42. Derivation of the equation: The rate of appearance of products ( i . e ., velocity, V) is proportional to the concentration of the enzyme-substrate complex. V α ES V = k (ES) -------------------- (1) The maximum reaction rate, V m will occur at a point where the total enzyme E t is bound to the substrate. V m α E t V m = k ( E t ) ----------------------(2)

43. Dividing equation (1) by (2,) we get : V = k (ES) --------------- V m = k (E t ) ------------ (3) Now coming back to the reversible reaction, E + S ES , one can write the equilibrium constant for dissociation of ES as K m which is equal to :

44. Michaelis-Menten equation

45. Michaelis-Menten plot This plot is used to determine the V m and K m value of the enzyme

46. Determination of V m and K m value When V = ½ V m K m = S

47. Significance of V m and K m value K m or Michaelis-Menten constant is defined as the substrate concentration (expressed in moles/l) to produce half-maximum velocity in an enzyme catalyzed reaction The K m values of the enzymes differ greatly from one to other, but it is a characteristic feature of a particular enzyme. for most of the enzymes, the general range is between 10 −1 and 10 −6 M

48. The K m value depends on the particular substrate and on the environmental conditions such as temperature and ionic concentration. But it is not dependent on the concentration of enzyme K m is a measure of the strength of ES complex. The high K m value indicates weak binding whereas the low K m value signifies strong binding. The maximal rate ( V m ) represents the turnover number of an enzyme, if the concentration of the active sites (Et) is known.

49. ENZYME INHIBITION Enzyme inhibitor is defined as a substance which binds with the enzyme and brings about a decrease in catalytic activity of that enzyme. Inhibitor may be organic or inorganic in nature . There are three broad categories of enzyme inhibition 1. Reversible inhibition . 2. Irreversible inhibition .

50. Reversible inhibition: The inhibitor binds non-covalently with enzyme Enzyme inhibition can be reversed if the inhibitor is removed. The reversible inhibition is further sub-divided into l . Competitive inhibition ll . Non-competitive inhibition

51. l. Competitive inhibition The rate of inhibition depends on: Concentration of substrate and inhibitor Affinity of inhibitor towards the enzyme The inhibition can be reversed by increasing the concentration of substrate

52. Km value increases whereas Vmax remains unchanged Eg ., succinate dehydrogenase Original substrate - succinic acid Inhibitor – malonic acid, glutaric acid, oxalic acid Competitive inhibitors have clinical significance

53. ll . Non-competitive inhibition The rate of inhibition depends on the concentration of the inhibitor Km remains constant whereas Vmax value decreases

54. Eg ., Various heavy metals ions (Ag + , Hg 2+ , Pb 2+ ) inhibit the activity of a variety of enzymes. Urease, for example, is highly sensitive to any of these ions in traces. Heavy metals form mercaptides with sulfhydryl (-SH ) groups of enzymes: cyanide and hydrogen sulfide strongly inhibit the action of iron-containing enzymes like catalase and peroxidase.

55. 2. Irreversible inhibition: The inhibitors bind covalently with the enzymes and inactivate them irreversibly These inhibitors are usually toxic poisonous substances Irreversible inhibitors combine with or destroy a functional group on the enzyme that is essential for its activity

56. Eg ., lodoacetate – irreversible inhibitor of papain and glyceraldehyde 3-phosphate dehydrogenase . Iodoacetate combines with sulfhydryl (-SH) groups at the active site of these enzvmes and makes them inactive Eg ., Diisopropyl fluorophosphate (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

57. Eg ., Organophosphorus insecticides like melathion are toxic to animals (including man ) as they block the activity of acetylcholine esterase (essential for nerve conduction ), resulting in paralysis of vital body functions Eg ., P enicillin antibiotics act as irreversible inhibitors of serine – containing enzymes, and block the bacterial cell wall synthesis

58. ENZYME REGULATION Covalent modification: Certain enzymes exist in the active and inactive forms which are interconvertible , depending on the needs of the body. The interconversion is brought about by the reversible covalent modifications, namely phosphorylation and dephosphorylation oxidation and reduction of disulfide bonds.

59. Covalent modification by phosphorylation-dephosphorylation of a seryl residue For some enzymes phosphorylation increases its activity whereas for some other enzymes it decreases the activity

60. Covalent modification by oxidation and reduction of disulfide bonds A few enzymes are active only with sulfhydryl (- SH) groups , Eg ., succinate dehydrogenase , urease. Substances like glutathione bring about the stability of these enzymes.

61. Allosteric regulation: They possess sites called allosteric site (other than that of active site) Certain substances referred to as allosteric modulators (effectors or modifiers ) bind at the allosteric site and regulate the enzyme activity . positive (+) allosteric effector – the binding of which increases the activity of the enzyme – so called as allosteric activator negative (-) allosteric effector – the binding of which decreases the activity of the enzyme – so called as allosteric inhibitor

62. Homotropic effect: modulator and substrate are same – mostly positive Heterotropic effect: modulator and substrate are different – may be positive or negative

63. Allosteric regulation mechanism