ENZYMES

ENZYME CLASSIFICATION, MECHANISM OF ACTION, SPECIFICITY & ACTIVE SITE.

1. Enzymes Gandham . Rajeev

2. Life is short and thus has to be catalyzed. Self replication and catalysis are believed to be the two fundamental conditions for life to be evolved

3. E nzymes HISTORY: Late 1700 – 1800 - Digestion of starch → sugar extracts in plants and saliva. Meat digestion by secretions in stomach were identified, but mechanism is unknown. In 19 th century - Fermentation of S ugar → alcohol in yeast, studied by Louis Pasteur In 1878, German physiologist Wilhelm Kühne first used the term enzyme, Greek "in living", to describe this process.

4.            The Nobel Prize in Chemistry 1946 “ For his discovery that enzymes can be crystallized" “ For their preparation of enzymes & virus proteins in a pure form"                                                      James Batcheller Sumner John Howard Northrop Wendell Meredith Stanley 1/2 of the prize 1/4 of the prize 1/4 of the prize Cornell University Ithaca, NY, USA Rockefeller Institute for Medical Research Princeton, NJ, USA Rockefeller Institute for Medical Research Princeton, NJ, USA 1887-1955 1891-1987 1904-1971

5. Leonor Michaelis (1875-1949 ) German Maud Menten (1879-1960 ) Canadian

6. Enzymes Organic bio catalysts - increase the rates of chemical Reactions. Accelerate reaction rate by a factor upto 10 6 or more Not consumed / altered by the reactions they catalyze. Highly powerful catalytic activity. Highly Specific in their action Thermolabile , colloidal in nature Most of the enzymes are Proteins in nature. Typical enzyme - Globular protein (62 – 2,500 A.A`s), M.wt - 12,000 to over 1 million .

7. Definition: Defined as organic biocatalysts synthesized by living cells. They are protein in nature (exception - RNA acting as ribozyme ), colloidal and thermolabile in character, and specific in their action . Enzyme catalysis is very rapid; usually 1 molecule of an enzyme can act upon about 1000 molecules of the substrate per minute. Lack of enzymes will lead to block in metabolic pathways causing inborn errors of metabolism.

8. Characteristics of Enzymes Almost all enzymes are proteins. Enzymes follow the physical and chemical reactions of proteins. They are heat labile. They are water-soluble. They can be precipitated by protein precipitating reagents (ammonium sulfate or trichloroacetic acid ). They contain 16% weight as nitrogen.

9. Ribozymes - RNAs with catalytic activity Play role in gene expression rather than metabolism. Site - in Cytoplasm, on a cell organelle, membrane bound, extracellular – interstitial or vascular space. In enzymatic reactions - Substrates - the molecules at the beginning of the process and Products - the enzyme converts them into different molecules at the end.

10. Biomedical Importance They determine the patterns of chemical transformations. They mediate the transformation of one form of energy into another. Deficiencies: In the quantity or catalytic activity of key enzymes - genetic /nutritional deficits, or toxins. Imbalances in enzyme activity - pharmacologic agents to inhibit specific enzymes. LIFE IS IMPOSSIBLE WITHOUT ENZYMES.

11. Naming of Enzymes According to the reaction they carry out. Suffix - ase is added to the name of the substrate ( e.g. , lactase is the enzyme that cleaves lactose) or the type of reaction e.g. , DNA polymerase forms DNA polymers). Systematic names – based on IUBMB - EC. International Union of biochemistry and molecular biology form system for nomenclature and classification

12. S pecificity of enzymes Enzymes are highly specific in their action Specificity is a characteristic property of the active site Types of enzyme specificity: Stereospecificity Reaction specificity Substrate specificity

13. Stereospecificity or optical specificity Stereoisomers are the compounds which have the same molecular formula, but differ in their structural configuration The enzymes act only on one isomer and, therefore, exhibit stereospecificity L-amino acid oxidase and D-amino acid oxidase act on L- and D-amino acids respectively.

14. Hexokinase acts on D-hexoses Glucokinase on D-glucose Amylase acts on α - glycosidic linkages Cellulase cleaves β - glycosidic bonds The class of enzymes belonging to isomerases do not exhibit stereospecificity , since they are specialized in the interconversion of isomers

15. Reaction specificity The same substrate can undergo different types of reactions, each catalysed by a separate enzyme and this is referred to as reaction specificity . An amino acid can undergo transamination, oxidative deamination , decarboxylation , racemization etc. The enzymes however, are different for each of these reactions.

16. Substrate specificity Absolute substrate specificity: Certain enzymes act only on one substrate e.g . glucokinase acts on glucose to give glucose 6 - phosphate , urease cleaves urea to ammonia and carbon dioxide Relative substrate specificity: Some enzymes act on structurally related substances, May be dependent on the specific group or a bond present . The action of trypsin is a good example for group specificity

17. Bond Specificity: Most of the proteolytic enzymes are showing group ( bond) specificity . E.g. trypsin can hydrolyse peptide bonds formed by carboxyl groups of arginine or lysine residues in any proteins Group Specificity: One enzyme can catalyse the same reaction on a group of structurally similar compounds, E.g. hexokinase can catalyse phosphorylation of glucose , galactose and mannose.

18. IUBMB classification of enzymes Based on the reaction they catalyze – grouped into 6 major classes - (OTHLIL) 1. Oxidoreductase 2. Transferase 3. Hydrolase 4. Lyase 5. Isomerase 6. Ligase

19. Oxidoreductases : This group of enzymes will catalyse oxidation of one substrate with simultaneous reduction of another substrate or co-enzyme. Catalyze oxidation/reduction reactions. They catalyze the addition of oxygen, transfer of hydrogen & transfer of electrons. AH 2 + B → A + BH 2 Subclasses: Oxidases & dehydrogenases

20. Oxidases Oxidases catalyse the transfer of hydrogen or electrons from donor, using oxygen as hydrogen acceptor - E.g. cytochrome oxidase Dehydrogenases: Dehydrogenases catalyse the transfer of hydrogen (or electrons), but the hydrogen acceptor is a molecule other than oxygen. The hydrogen acceptors are usually NAD or NADP & FAD or FMN - E.g. LDH

21. Oxido-reductases

22. 2. Transferases : This class of enzymes transfers one group ( other than hydrogen) from the substrate to another substrate. Transfer a functional group ( e.g. a methyl, alcoholic, aldehyde, ketone, acyl, sulphur or phosphate group). A–X + B → A + B–X Subclass: Transferases (amino transaminases) - amino group Kinases - phosphate group

23. Aminotransferases (transaminases): Catalyse the transfer of an amino group from one amino acid to an alpha ketoacid , resulting in the formation of new amino acid & new ketoacid E.g. AST Transaminases are clinically important. Kinases: Catalyse the transfer of phosphate from ATP (or GTP) to a substrate E.g. glucokinase

24. Transaminases

25. 3. Hydrolases: This class of enzymes can hydrolyse ester, ether , peptide or glycosidic bonds by adding water and then breaking the bond. Catalyze the hydrolysis of various bonds, like C-C, C-O, C-N, P-O and acid anhydride bonds. Phosphatases, Esterases , Peptidases, Lipases A–B + H 2 O → A–OH + B–H

26. Subclass Glycosidases & phosphatases Glycosidases catalyse the hydrolysis of glycosidic bonds E.g. maltase Phosphatases catalyse the removal of phosphate from substrate. E.g. glucose 6-phosphatase,

27. 4. Lyases : These enzymes can remove groups from substrates or break bonds by mechanisms other than hydrolysis to form double bonds and addition of groups to break double bonds. Addition or removal of groups to form double bonds. Catalyze cleavage of C-C, C-O, C-N and other bonds by elimination - Elimination and addition reactions. Decarboxylases, Synthases A -B + X-Y → AX - BY

28. Subclass: Lyases & Decarboxylases Lyases catalyse the cleavage of C-C bonds. E.g. citrate lyase Decarboxylases catalyse the release of CO 2 from the substrate such as alpha ketoacids & amino acids. E.g. Glutamate decarboxylase

29. 5. Isomerases : Catalyze intra-molecular group transfer (transfer of groups within the same molecule). These enzymes can produce optical, geometric or positional isomers of substrates. E.g. Epimerases , Mutases , Racemases , epimerases , cis -trans isomerases Interconversion of isomers. A → A'

30. Subclass: Isomerases & epimerases Isomerases catalyse the interconversion of cis -trans isomers & functional isomers E.g. Phosphohexoseisomerase . Epimerases catalyse the interconversion of epimers . E.g. Phosphopentose epimerase

31. 6. Ligases: These enzymes link two substrates together , usually with the simultaneous hydrolysis of ATP , ( Latin, Ligare = to bind). Catalyze formation of C-C, C-S , C-O, or C-N bonds, by condensation reactions, involving ATP. A-OH + B-H A-B

32. Subclass: Carboxylases & syntheteses Carboxylases catalyse the formation of C-C bonds using CO 2 (HCO 3 ) as substrate. E.g. Pyruvate carboxylase S yntheteses are enzymes that link two molecules with covalent bonds in ATP dependent reaction. E.g. Glutamine synthetase

33. Ligases ATP →ADP + Pi

34. IUBMB - EC numbers Each enzyme is described by a sequence of four numbers preceded by "EC " First digit represents the class - classifies the enzyme based on its reaction. Second digit stands for the subclass - indicates the type of group involved in the reaction. Third digit is the sub-subclass or subgroup - indicates substrate on which group acts. Fourth digit gives the number of the particular enzyme in the list- indicates - serial number of individual enzyme.

35. Lactate dehydrogenase ( lactate:NAD + oxidoreductase )

36. Enzyme Nomenclature and Classification EC Classification Class Subclass Sub-subclass Serial number

37. Classification Based on where enzyme activity occurs – Exoenzymes - Digestive enzymes (pepsin, sucrase ) Endoenzymes - endopeptidases

38. Classification based on complexity Simple, monomeric enzymes Digestive enzymes (pepsin, sucrase ) Multimeric >1 protein chain, >1 active site Multienzyme complexes Aggregates of a number of different enzymes All enzymes in complex catalyze series of related reactions E.g. FAS complex, PDH complex. etc.

39. Co-enzymes Enzymes may be simple proteins, or complex enzymes , containing a non-protein part, called the prosthetic group. The prosthetic group is called the co-enzyme. It is heat stable . Salient features of co-enzymes: The protein part of the enzyme gives the necessary three dimensional infrastructure for chemical reaction ; but the group is transferred from or accepted by the co-enzyme

40. Essential for the biological activity of the enzyme It is a low molecular weight organic substance The co-enzymes combine loosely with the enzyme molecules & separated easily by dialysis When the reaction is completed, the co-enzyme is released from the apo -enzyme, and can bind to another enzyme molecule One molecule of the co-enzyme is able to convert a large number of substrate molecules with the help of enzyme Most of them are derivatives of B complex vitamin

41. (Vitamins)

42. Non – Vitamin Coenzymes ATP Donates Phosphate , adenosine , AMP moieties CDP Required in phospholipid synthesis as a carrier of choline, ethanolamine UDP Carrier of glucose – glycogen synthesis galactose SAM Methyl group donor PAPS Sulfate group donor in mucopolysaccharide synthesis

43. Cofactors Enzymes may be simple proteins or Compound. Many enzymes require small molecules or metal ions to participate directly in substrate binding or catalysis. Active enzyme / Holoenzyme . Polypeptide portion of enzyme ( apoenzyme ) Nonprotein prosthetic group (cofactor) They can be - inorganic metal ions - cofactors or activators. complex organic or metallo -organic – coenzymes Cofactors are bound to the enzyme to maintain the correct configuration of the active site .

44. Prosthetic groups: Some cofactors bind to the enzyme protein very tightly (non-covalently or covalently). e.g – FMN , PLP, Biotin, Cu , Mg, Zn Metalloenzymes : Enzymes with tightly bound metal ions. Some metal ions (Fe 2 + , Cu 2 + ) participate in redox reactions. Others stabilize either the enzyme or substrate over the course of the reaction.

45. Metal-activated enzymes - Enzymes that require a metal ion cofactor. Apoenzyme + cofactor = Holoenzyme A holoenzyme also refers to the assembled form of a multiple subunit protein. Holoenzyme : A complete, catalytically active enzyme together with its bound cofactors.

46. Certain Vitamins - act as precursors of coenzymes. Coenzymes usually function as transient carriers of specific functional groups -Substrate Shuttles. Coenzyme stabilizes unstable substrates such as H atoms or hydride ions in the aqueous environment of the cell.

47. Second Substrates - Since coenzymes are chemically changed as a consequence of enzyme action, they are also named so. Common to many different enzymes - about 700 enzymes are known to use the coenzyme NADH. Coenzymes are usually regenerated and their concentrations maintained at a steady level inside the cell. e.g - NADPH is regenerated through the pentose phosphate pathway & S- adenosylmethionine by methionine adenosyltransferase .

48. Figure 5.3

49. Mechanism of Enzyme Action Catalysis is the prime function of enzymes F or any chemical reaction to occur , the reactants have to be in an activated state or transition state . Generation of transition state complexes & formation of products: Binding of the substrate to the active site of the enzyme causes bonding rearrangements that leads to an intermediate state called “transition-complex”

50. This is an activated form of substrate immediately preceding the formation of products. An enzyme speeds a reaction by lowering the activation energy Less energy is needed to convert reactants to products. This allows more molecules to form product. Activation free energy (G): The energy required to convert substrates from ground state to transition state.

51. Substrates need a large amount of energy to reach a transition state, which then decays into products. The enzyme stabilizes the transition state , reducing the energy needed to form products The enzyme does not affect the equilibrium position of the reaction

52. Enzyme-Substrate Binding

53. Steps of Enzyme Catalysis Formation of enzyme – substrate complex . Generation of Transition-state complexes Formation of Reaction Products

54. ES Complex

55. ES Complex

56. Theories to explain ES Complex Lock and key model or Fischer's template theory The active site has a rigid shape. Only substrates with the matching shape can fit. The substrate is a key that fits the lock of the active site. Fails to explain the stabilization of the transition state, action of allosteric modulators.

57. Active site of unbound enzyme is complementary in shape to substrate

58. Induced-fit Model The active sites of some enzymes assume a shape that is complementary to that of the transition state only after the substrate is bound. The active site is flexible, not rigid. Substrate binding brings conformation changes in active site – nascent active site Enables strong binding site - improves catalysis. There is a greater range of substrate specificity.

59. Active site forms a shape complementary to substrate only after it is bound

60. Substrate strain theory As the substrate flexes to fit the active site, bonds in the substrate are flexed and stressed. This causes changes/conversion to product. Induced fit and substrate strain combinedly operate in enzyme action.

61. Mechanism of enzyme catalysis The formation of an enzyme-substrate complex (ES ) is very crucial for the catalysis to occur , and for the product formation. It is estimated that an enzyme catalysed reaction proceeds 106 to 1012 times faster than a non-catalysed reaction The enhancement in the rate of the reaction is mainly due to four processes: Acid-base catalysis Substrates train Covalent catalysis Entropy effects

62. Acid-base catalysis Role of acids and bases is quite important in enzymology. At the physiological pH , histidine is the most important amino acid, the protonated form of which functions as an acid and its corresponding conjugate as a base. The other acids are –OH group of tyrosine, -SH group of cysteine, and e-amino group of lysine . The conjugates of these acids and carboxyl ions (COO-) function as bases. Ribonuclease which cleaves phosphodiester bonds in a pyrimidine loci in RNA is a classical example of the role of acid and base in the catalysis

63. Substrate strain During the course of strain induction, the energy level of the substrate is raised, leading to a transition state . The mechanism of lysozyme (an enzyme of tears , that cleaves β -1,4 glycosidic bonds) action is believed to be due to a combination of substrates strain and acid-base catalysis

64. Covalent catalysis In the covalent catalysis , the negatively charged ( nucleophilic ) or positively charged ( electrophilic) group is present at the active site of the enzyme. This group attacks the substrate that results in the covalent binding of the substrate to the enzyme. In the serine proteases (so named due to the presence of serine at active site), covalent catalysis along with acid-base catalysis occur , e.g . chymotrypsin, trypsin etc

65. Entropy effect Entropy is a term used in thermodynamics. I t is defined as the extent of disorder in a system The enzymes bring about a decrease in the entropy of the reactants. This enables the reactants to come closer to the enzyme and thus increase the rate of reaction. In the actual catalysis of the enzymes , more than one of the processes acid-base catalysis, substrate strain, covalent catalysis and entropy are simultaneously operative. This will help the substrate (s) to attain a transition state leading to the formation of products.

66. T hermodynamics of enzymatic reactions The enzyme catalysed reactions may be broadly grouped into three types based on thermodynamic (energy) considerations. lsothermic reactions: The energy exchange between reactants and products is negligible. e.g . glycogen phosphorylase Glycogen + Pi Glucose 1-phosphate

67. Exothermic (exergonic) reactions: Energy is liberated in these reactions . E.g. urease Endothermic (endergonic) reactions: Energy is consumed in these reactions e.g . glucokinase Glucose + ATP Glucose 6-phosphate + ADP Urea NH 3 + CO 2 + energy

68. Active site The active site (or active centre) of an enzyme represents as the small region at which the substrate(s ) binds and participates in the catalysis Active site is due to tertiary structure of protein. Clefts / crevices – provide suitable environment for reaction

69. Salient features of active site The existence of active site is due to the tertiary structure of protein resulting in three dimensional native conformation The active site is made up of amino acids ( known as catalytic residues ) which are far from each other in the linear sequence of amino acids ( primary structure of protein). For instance , the enzyme lysozyme has 129 amino acids.

70. Lysozyme has 129 amino acids. The active site is formed by the contribution of amino acid residues numbered - 35, 52, 62, 63 and 101. 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 in structure and shape. It is rather flexible to promote the specific substrate binding .

71. The active site possesses a substrate binding site and a catalytic site. The latter is for the catalysis of the specific reaction . The coenzymes or cofactors on which some enzymes depend are present as a part of the catalytic site . The substrate ( s) binds at the active site by weak non-covalent bonds . Enzymes are specific in their function due to the existence of active sites.

72. The commonly found amino acids at the active sites are serine, aspartate, histidine , cysteine, lysine, arginine, glutamate, tyrosine. Among these amino acids, serine is the most frequently found. The substrate (s) 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

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