Enzymes by pabitra subedi / sanjiv .pptx

enzyme

1. ENZYMES PABITA SUBEDI

2. INTRODUCTION Enzymes are colloidal, organic macromolecules which are produced in the living organisms and catalyze biochemical reactions without being utilized in the process. Enzymes enhance the rates of the corresponding not-catalyzed reaction by factors of at least 10 6 . Biocatalysts are designated as enzyme since they were discovered as the substances found in yeasts (Greek- en=in, and zyme =yeast). A good teacher is always a good catalyst in students’ life!

3. DIFFERENCE BETWEEN AN ENZYME AND A CHEMICAL CATALYST Enzymes Inorganic catalyst 1. These are synthesized by living cells. These are not produced by living cells. 2. These are very delicate and tend to denature above 40°C temperature. Most of them are maximally active at 37 °C. These are not heat sensitive and can be heated well above 100 °C without altering their catalytic activity. 3. Each enzyme is highly specific and requires specific substrate and majority of them carry out only one type of reaction. These are capable of carrying out wide range of chemical reactions. 4. These are sensitive to pH changes and also to the presence of metallic activators. These are not sensitive to pH changes and to the presence of metallic activators. 5. Heavy metals denature the enzymes. Heavy metals do not affect the catalytic activity of the inorganic catalysts.

4. GENERAL PROPERTIES The important properties of enzymes are: Chemical nature Almost all enzymes are proteins (exception – RNA acting as ribozyme ). Because of their protein nature, enzymes are subjected to denaturation at high temperature, low pH or high pH. Catalytic efficiency Enzyme catalyzed reactions are highly efficient, proceeding from 10 3 -10 8 times faster than uncatalyzed reactions. Enzyme specificity Enzymes are highly specific, interacting with one or a few substrates and catalyzing only one type of chemical reaction. (the set of enzymes made in cell determines which metabolic pathways occur in that cell).

5. Enzyme regulation Enzyme activity can be regulated, that is, increased or decreased, so that the rate of product formation responds to cellular need. Active sites Enzyme molecules contain a special pocket or cleft called the active site. The active site contains amino acid side chains that participate in substrate binding and catalysis. Location within the cell Many enzymes are localized in specific organelles within the cell. Such compartmentalization serves to isolate the reaction substrate or product from other competing reactions. This provides a favourable environment for the reaction, and organizes the thousands of enzymes present in the cell into purposeful pathways. Holoenzymes Some enzymes require molecules other than proteins for enzyme activity. The term holoenzyme refers to the active enzyme with its nonprotein component, whereas the enzyme without its nonprotein moiety is termed an apoenzyme and is inactive.

6. HOLOENZYME The functional unit of the enzyme is known as holoenzyme which is made up of apoenzyme (the protein part) and a coenzyme (non-protein organic part). Holoenzyme Apoenzyme + Coenzyme ( Active enzyme) ( protein part ) (non-protein part) The term prosthetic group is used when the non-protein moiety tightly (covalently) binds with the apoenzyme . The coenzyme can be separated by dialysis from the enzyme while the prosthetic group cannot be. Some of the enzymes require the presence of certain molecules, such as a metal ion or an organic molecule for their activity. The inorganic ions, such as Mg 2+ , Zn 2+ or Cl - required for the catalytic activity of an enzyme are called as cofactors. Fig. Holoenzyme

7. CO-ENZYME Definition The non-protein, organic, low molecular weight, heat stable and dialysable substance associated with enzyme function is known as coenzyme . Salient features of co-enzymes Coenzymes are often regarded as the second substrates or co-substrates, since they have affinity with the enzyme comparable with that of the substrates. 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 coenzyme . The co-enzyme is essential for the biological activity of the enzyme. Generally, the co-enzymes combine loosely with the enzyme molecules. The enzyme and co-enzyme can be separated easily by dialysis. Inside the body, 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 the co-enzymes are derivatives of vitamin B complex substances.

8. Coenzymes from B-complex vitamins Most of the coenzymes are the derivatives of water soluble B-complex vitamins. Coenzyme (abbreviation) Derived from vitamin Atom or group transferred Dependent enzyme (example) Thiamine pyrophosphate (TPP) Thiamine Aldehyde or keto Transketolase Flavin mononucleotide (FMN) Riboflavin Hydrogen and electron L-amino acid oxidase Flavin adenine dinucleotide (FAD) Riboflavin Hydrogen and electron D-amino acid oxidase Nicotinamide adenine dinucleotide (NAD + ) Niacin Hydrogen and electron Lactate dehydrogenase Nicotinamide adenine dinucleotide phosphate (NADP+) Niacin Hydrogen and electron Glucose 6-phosphate dehydrogenase Lipoic acid Lipoic acid Hydrogen and electron Pyruvate dehydrogenase complex

9. Non-vitamin coenzymes Not all coenzymes are vitamin derivatives. There are some other organic substances, which have no relation with vitamins but function as coenzymes. They may be considered as non-vitamin coenzymes. Coenzymes not related to B-complex vitamins Coenzyme Abbreviation Biochemical functions Adenosine triphosphate ATP Donates phosphate, adenosine and adenosine monophosphate (AMP) moieties. Cytidine diphosphate CDP Required in phospholipid synthesis as carrier of choline and ethanolamine. Uridine diphosphate UDP Carrier of monosaccharide (glucose, galactose), required for glycogen synthesis S- Adenosylmethionine (active methionine) SAM Donates methyl group in biosynthetic reactions Phosphoadenosine phosphosulfate ( acitve sulfate ) PAPS Donates sulfate for the synthesis of mucopolysaccharides .

10. Nucleotide coenzymes Some of the coenzymes possess nitrogenous base, sugar and phosphate, such coenzymes are, therefore, regarded as nucleotides e.g. NAD+, NADP+, FMN, FAD, coenzyme A etc. Protein coenzymes Thioredoxin is a protein that serves as a coenzyme for the enzyme ribonucleotide reductase.

11. LOCALIZATION OF ENZYMES Intracellular enzymes E nzymes which are produced by the cells of a particular tissue and function within the cell. Such enzymes are called as intracellular enzymes, e.g. the enzymes of glycolysis, TCA cycle and fatty acid synthesis . Extracellular enzymes Enzymes which are produced by the cells of a particular tissue and liberated from there for use in the other tissues. Such enzymes are called as extracellular enzymes, e.g. various proteolytic enzymes like trypsin, chymotrypsin etc. They are secreated by the pancreatic juice for their action in the small intestine. Fig. I ntracellular localization of some important biochemical pathways.

12. ZYMOGEN Most of the intracellular enzymes are secreated in their active form, called ‘ zymase ’ form of the enzyme. Proteolytic enzymes are usually synthesized as somewhat larger inactive precursors, known as ‘zymogens ’. Zymogens are stored in the storage vesicles known as zymogen granules , secreted as zymogens and undergo modifications in structure, after coming in contact with certain activating agents.

13. Gastric and pancreatic Proenzymes /Zymogens Proenzyme / Zymogen Site of synthesis Activator Active enzyme Pepsinogen Chief cells of stomach H+, pepsin Pepsin Trypsinogen Pancreatic acinar cells Trypsin Trypsin Chymotrypsinogen Pancreatic acinar cells Trypsin ∏ - or α -chymotrypsin Procaroxypeptidase A Pancreatic acinar cells Trypsin Carboxypeptidase A Procarboxypeptidase B Pancreatic acinar cells Trypsin Carboxypeptidase B Proelastae Pancreatic acinar cells Trypsin Elastase

14. The inactivity of zymogens is crucial because if these enzymes were synthesized in their active forms within the cell, this situation would be potentially self-destructing. Acute pancreatitis, a painful and sometimes fatal condition is characterized by the premature activation of the digestive enzymes synthesized by this organ. The enzymes damage the pancreas and spill over into the circulation. Pancreatic amylase is found at very high concentration in the serum of these patients and helps to establish the diagnosis. ACUTE PANCREATITIS

15. Each enzyme is assigned two names. The first is its short, recommended name, convenient for everyday use. The second is the more complete systematic name, which is used when an enzyme must be identified without ambiguity. Recommended name Most commonly used enzyme names have the suffix “- ase ” attached to the substrate of the reaction (for example, lipase, fatty acid synthase and urease), or to a description of the action performed (for example, lactate dehydrogenase and adenylyl cyclase ). These names of the enzymes are called as trivial names. CLASSIFICATION OF ENZYMES

16. Systematic name To standardize enzyme nomenclature, the Enzyme Commission (EC) of the IUB (international Union of Biochemistry) adopted a classification system in 1961. The IUB system assigns a systematic name to each enzyme, defining the substrate acted on, the reaction catalyzed, and possibly, the name of any coenzyme involved in the reaction. The systematic names are unambiguous and informative, but are frequently too cumbersome to be of general use, therefore, a more usable, trivial, recommended name is also assigned by the IUB system. In the systematic name system, enzymes are divided into six major classes. The word OTHLIL (first letter in each class) may be memorised to remember the six classes of enzymes in the correct order). Each major class is divided into subclasses. Subclasses are further divided into subsubclasses .

17. Enzyme commission code number Each enzyme is assigned a code – four digit code number, with each digit separated from the next by a period. First digit places the enzyme in one of the six classes. Second digit defines the subclass defining specific functional characteristics. Third digit defines sub-subclass indicating the nature or reaction. Fourth digit defines the specific substrate.

18. FACTORS INFLUENCING ENZYME ACTION Enzyme concentration Rate of a reaction or velocity (V) is directly proportional to the enzyme concentration, when sufficient substrate is present. This property is made use of determining the level of particular enzyme in plasma, serum or tissues for the diagnosis of diseases. Known volume of serum is incubated with substrate for a fixed time, then reaction is stopped and product is quantitated . Since the product formed will be proportional to the enzyme concentration, the latter could be assayed.

19. 2. 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).

20. 3. Effect of temperature Velocity of enzyme reaction increases with increase in temperature up to a maximum and then declines (Bell shaped curve). The temperature at which maximum amount of the substrate is converted to the product is called optimum temperature. The optimum temperature for most human enzymes is between 35 and 40°C. Human enzymes start to denature at temperature above 40°C, but thermophilic bacteria found in the hot springs have optimum temperatures of 70 °C. The reaction velocity of most chemical reactions increase with temperature approximately double for each 10°C rise called temperature coefficient Q 10 . A bell-shaped curve is usually observed. Clinical significance Foods can be preserved in refrigerators (at low temperatures) due to reduced bacterial enzyme activities. Certain surgeries are carried out by lowering the patient’s body temperature (induced hypothermia), and thus the metabolic rate.

21. 4. Effect of pH Each enzyme has an optimum pH, on both sides of which the velocity will be drastically reduced. The relationship between enzyme activity and pH is represented by a bell shaped curve which has its peak at the optimum pH. Usually enzymes have the optimum pH between 6 and 8. The optimum pH for pepsin however, is about 2.0 while for the enzymes of the pancreatic juice, optimum pH is nearly 8.0, for alkaline phosphatases is around 10. This pH is characteristic for each enzyme.

22. 5. Effect of Product concentration The accumulation of reaction products generally decreases the enzyme velocity . 6. Effect of activators Some of the enzymes require certain inorganic metallic cations like Mg 2+ , Mn 2+ , Zn 2+ etc. for their optimum activity. Rarely, anions are also needed for enzyme activity e.g. Chloride ion ( Cl - ) for amylase. Metals function as activators of enzyme velocity through various mechanisms- combining with the substrate, formation of ES-metal complex etc . 7. Effect of light and radiation Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivates certain enzymes due to the formation of peroxides. E.g. UV rays inhibit salivary amylase activity.

23. ENZYME KINETICS The study of reaction rates and how they change in response to changes in experimental parameters is known as enzyme kinetics . One of the key factors affecting the rate of a reaction catalyzed by an enzyme is the amount of substrate present [S]. The effect on V (initial velocity) of varying substrate [S] concentration, when enzyme concentration is held constant, is shown in fig. At relatively low concentration of substrate, V increases almost linearly with an increase in [S], a condition known as first order kinetics. At higher substrate concentration, V increases by smaller and smaller amounts in response to increase in [S]. Finally, a part is reached beyond which there are only vanishingly small increase in V with increasing [S], a condition known as zero order kinetics and a plateau is called maximum velocity, V max . Fig. Effect of substrate concentration on reaction velocity for an enzyme catalyzed reaction

24. The ES complex is the key to understanding the kinetic behaviour. The kinetic pattern led Victor Henri to propose in 1903 that the combination of an enzyme with its substrate molecule to form an enzyme substrate complex is a necessary step in enzyme catalysis. This idea was expanded into a general theory of enzyme action, particularly by Leonor Michaelis and Maud Menten in 1913. They postulated that the enzyme first combines reversibly with its substrate to form an enzyme-substrate complex in a relatively fast reversible step: E + S ES The ES complex then breaks down in a slower second step to yield the free enzyme and the reaction product P: ES E + P Because the slower second reaction must limit the rate of the overall reaction, the overall rate must be proportional to the concentration of the species that reacts in the second step, that is, ES. K 2 K1 K -1 K -2

25. At any given instant, in an enzyme catalyzed reaction, the enzyme exists in two forms, the free form or uncombined form E and the combined form ES. At low [S], most of the enzyme will be in the uncombined form E. Here, the rate will be proportional to [S]. The maximum velocity ( V max ) of the catalyzed reaction is observed when virtually all of the enzymes are present as the ES complex and concentration of E is vanishingly small. Under these conditions, the enzyme is saturated with its substrate, and all the free enzymes will have been converted into ES form. So that any further increase in [S] has no effect on the rate and the reaction quickly achieves a steady state, in which [ES] remains approximately constant over the time to form a plateau.

26. MICHAELIS-MENTEN EQUATION The Michaelis-Menten equation describes how reaction velocity varies with substrate concentration. V = V max [S] K m +[S] Where V is the initial velocity V max is the maximal velocity K m is the Michaelis constant = ( k -1 + k 2 )/k 1 [S] is the substrate concentration

27. The kinetic curves expressing the relationship between V and [S] have the same general shape (a rectangular hyperbola) for most enzymes, which can be expressed algebraically by the MM equation. Michaelis and Menten derived this equation starting from their basic hypothesis that the rate-limiting step in enzymatic reactions is the breakdown of the ES complex to product and free enzyme. The MM equation is V = V max [S]/(K m + [S]). All these terms, [S], V , V max , as well as the constant called the Michaelis constant, K m , can be readily measured experimentally. The derivation of the MM equation starts with the two basic steps of the formation and breakdown of ES. Early in the reaction, the concentration of the product [P] is negligible, and a simplifying assumption is made that the reaction P  S (described by k -2 ) can be ignored. The overall reaction then reduces to k 1 k 2 E + S ⇄ ES  E + P. k -1 V is determined by the breakdown of ES to form product, which is determined by [ES] through the equation V = k 2 [ES]. Because [ES] in the above equation is not easily measured experimentally, an alternative expression for this term must be found. First, the term [E t ], representing the total enzyme concentration (the sum of free and substrate-bound enzyme) is introduced. Free or unbound enzyme [E] can then be represented by [E t ] - [ES]. Also, because [S] is ordinarily far greater than [E t ], the amount of substrate bound by the enzyme at any given time is negligible compared with the total [S]. With these conditions in mind, the following steps lead to an expression for V in terms of easily measurable parameters. DERIVATION OF MICHAELIS-MENTEN EQUATION

28. Derivation of the MM Equation

29. Derivation of the MM Equation

30. Derivation of the MM Equation The MM equation describes the kinetic behavior of a great many enzymes, and all enzymes that exhibit a hyperbolic dependence of V on [S] are said to follow Michaelis-Menten kinetics. However the MM equation does not depend on the relatively simple two-step reaction mechanism discussed above. Many enzymes that follow MM kinetics have quite different mechanisms, and enzymes that catalyze reactions with six or eight identifiable steps often exhibit the same steady-state kinetic behavior. Even though the MM equation holds true for many enzymes, both the magnitude and the real meaning of V max and K m can differ from one enzyme to another. This is an important limitation of the steady-state approach to enzyme kinetics.

31. Validation of the MM Equation The MM equation can be shown to correctly explain the V vs [S] curves of many enzymes by considering limiting situations where [S] is very high or very low (Fig. 6-12). At low [S], K m >> [S] and the [S] term in the denominator of the MM equation becomes insignificant. The equation simplifies to V = V max [S]/K m and V exhibits a linear dependence on [S], as is observed at the left side of V vs [S] graphs. At high [S], where [S] >> K m , the K m term in the denominator of the MM equation becomes insignificant and the equation simplifies to V = V max . This is consistent with the plateau in V observed at high [S] in kinetic graphs. An important numerical relationship emerges from the MM equation in the special case when V is exactly one-half V max . Here V max /2 = V max [S]/(K m + [S]). On dividing by V max , the equation is 1/2 = [S]/(K m + [S]). After solving for K m , we get K m + [S] = 2[S], or K m = [S]. This is a very useful, practical definition of K m . K m is equivalent to the substrate concentration at which V is one-half V max .

32. Double-reciprocal Plots Because the plot of V vs [S] for an enzyme-catalyzed reaction asymptotically approaches the value of V max at high [S], it is difficult to accurately determine V max (and thereby, K m ) from such graphs. The problem is readily solved by converting the Michaelis-Menten kinetic equation to the so-called double-reciprocal equation ( Lineweaver -Burk equation) which describes a linear plot from which V max and K m can be easily obtained (Box 6-1, Fig. 1). The Lineweaver -Burk equation is derived by first taking the reciprocal of both sides of the Michaelis-Menten equation 1/V = (K m + [S])/ V max [S] Separating the components of the numerator on the right side of the equation gives 1/V = K m / V max [S] + [S]/ V max [S] Which simplifies to 1/V = K m / V max [S] + 1/ V max . The plot of 1/V vs 1/[S] gives a straight line, the y-intercept of which is 1/ V max and the x-intercept of which is -1/K m .

33. MICHAELIS-MENTEN CONSTANT(Km) Definition It is defined as the substrate concentration (expressed in moles/l) to produce half-maximum velocity in an enzyme catalyzed reaction. Salient Features of Km 1. Km value is substrate concentration (expressed in moles/L) at half-maximal velocity. 2. It denotes that 50% of enzyme molecules are bound with substrate molecules at that particular substrate concentration. 3. Km is independent of enzyme concentration. If enzyme concentration is doubled, the V max 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. 4. 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. 5. 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. 6. For majority of the enzymes, the Km value are in the range of 10-5 to 10-2 .

34. SIGNIFICANCE OF Km Physiological significance Glucose can be phosphorylated to glucose 6-phosphate by glucokinase (the enzyme present in the liver, is specific for glucose) or hexokinase (nonspecific for hexoses ) except galactose, present in all tissues). Glucokinase has a high Km (i.e. low affinity for glucose ) hence it is important during the fed state when glucose is in excess. Under post-absorptive/fasting conditions, hexokinase having a low Km (i.e. high affinity for glucose) is important so that glycolysis continues to provide energy to vital organs even at low blood glucose levels. 2. Laboratory significance During enzyme assay in the laboratory, the substrate concentration is kept at saturating amounts (at least 10 times the Km) so that the reaction proceeds to completion . 3. Clinical significance The Km value for a given enzyme may differ person to person and explains the varied response to drugs/chemicals. Aldehyde dehydrogenase enzyme oxidizes acetaldehyde (formed from alcohol) into acetic acid. People having a low Km (i.e. high affinity) variant of the enzyme metabolize acetaldehyde rapidly and are less susceptible to its adverse effects such as headache and flushing. On the other hand, having the high Km variant are more prone to these side effects of alcohol.

35. Km for Some Enzymes and Substrates Enzyme Substrate Km ( mM ) Hexokinase (brain) ATP D-Glucose D-Fructose 0.4 0.05 1.5 Carbonic anhydrase HCO3 - 26 Chymotrypsin Glycyltyrosinylglycine N- Benzoyltyrosinamide 108 2.5 β - Galactosidase D-Lactose 4.0 Threonine dehydratase L-Threonine 5.0

36. Lineweaver -Burk plot It is the transformed form of Michael- Menten equation. V = V max [S] Km + [S] The Michel- Menten equation can be algebraically transformed by taking the reciprocal of both sides of the Michaelis-Menten equation. 1 = Km + [S] V V max [S]

37. Separating the components of the numerator on the right side of the equation gives 1 = Km × 1 + [S] V V max [S] V max [S] 1 = Km + 1 V V max [S] V max This form of the Michaelis-Menten equation is called the Lineweaver -Burk equation.

38. For enzymes obeying the Michaelis-Menten relationship, a plot of 1/ V verses 1/ [S] yields a straight line. The double-reciprocal presentation, also called a Lineweaver -Burk plot, has the great advantage of allowing a more accurate determination of Vmax , which can only be approximated from a simple plot of V verses [S]. The double-reciprocal plot of enzyme is very useful in distinguishing between certain types of enzymatic reaction mechanisms and in analyzing enzyme inhibition.

39. MECHANISM OF ENZYME ACTION Similar to the requirement of energy for doing any physical work, all chemical reactions have a potential energy barrier. The energy required to convert all molecules of a reacting substance from the ground state to the transition state is known as activation energy. The transition state corresponds to the point of highest free energy. The difference in free energy between the reactants and the transition state is known as the free energy of activation (∆G°). An enzyme acts by lowering the transition state free energy for the reaction it catalyzes. For example, activation energy for acid hydrolysis of sucrose is 26,000 cal/mol, while the activation energy is only 9,000 cal/mol when hydrolyzed by sucrase. Fig. Enzymes lower the activation energy

40. Enzyme-Substrate C omplex F ormation Formation of enzyme-substrate (ES) complex is the first step in enzymatic catalysis which ultimately results in the product formation(p). E + S ES E + P Two models for substrate binding to the active site of the enzyme have been proposed to explain the specificity that an enzyme has for its substrate. Lock and Key model or Rigid Template Model of Emil Fisher Induced Fit Model or Hand-in-glove Model of Daniel E Koshland Lock and key Model First model purposed to explain an enzyme catalyzed reaction. According to this model, the structure of conformation of the enzyme is rigid. The substrate fits to the binding site (now active site) just as a key fits into the proper lock. This model failed because it does not explain the changes in the enzyme activity in the presence of allosteric modulators and also does not give any scope for the flexible nature of the enzymes.

41. Fig. Fischer’s template theory

42. Induced Fit Model Daniel E Koshland in 1958 postulated that the enzymes are flexible and shapes of the active site can be modified by the binding of the substrate. In the induced fit model, the substrate induces a conformational change in the enzyme, in the same manner in which placing a hand (substrate) into a glove (enzyme) induces changes in the glove’s shape. Therefore, this model is also known as hand-in-glove model. The functional groups of the active sites are arranged in a definite spatial configuration and the enzyme-substrate complex is formed by multiple bindings (such as by covalent bonds, hydrogen bonds and electrostatic bonds) of the substrate with the enzyme. When substrate analog is fixed to the enzyme, some structural alteration may occur, but reaction does not take place due to lack of proper alignment. Action of allosteric modulators and competitive inhibition can also be explained by the hypothesis of Koshland’s model.

43. Fig. Koshland’s induced fit theory

44. ENZYME ACTIVATORS In presence of certain inorganic ions, some enzymes show higher activity. Thus, chloride ions activate salivary amylase and calcium ions activate lipase. Another type of activation is the conversion of an inactive pro-enzyme or zymogen to the active enzyme. By splitting a single peptide bond, and removal of a small polypeptide from trypsinogen, the active trypsin is formed. This results in unmasking of the active center. Similarly trypsin activates chymotrypsinogen , so that A peptide (1-13 amino acids), B peptide (16-46) and C peptide (149-245) are formed from chymotrypsinogen . These 3 segments align in such a manner that histidine (57) and aspartate (102) and serine (195) residues form the active site.

45. ENZYME INHIBITION INTRODUCTION Chemical substances which inhibit enzyme activity and reduce the velocity of an enzyme catalyzed reaction are called inhibitors , e.g. cyanide inhibits cytochrome oxidase. The phenomenon of a decrease in enzymatic reaction brought about by the addition of an inhibitor is called enzyme inhibition . TYPES Competitive inhibition Non-competitive inhibition

46. Competitive inhibition (Reversible) A substance that competes directly with a normal substrate for an enzyme’s substrate-binding site is known as a competitive inhibitor. Chemical structure of the competitive inhibitor closely resembles with that of the substrate. They are called structural analogs. The inhibitor forms a complex with the enzyme called as enzyme-inhibitor complex (EI), instead of the enzyme substrate complex (ES). A competitive inhibitor thus reduces concentration of free enzyme available for the substrate binding. The relative concentration of the substrate and inhibitor and their respective affinity with the enzyme determines the degree of competitive inhibition. The degree of inhibition can be reduced by increasing the concentration of the substrate. Competitive inhibition is usually reversible or excess substrate abolishes the inhibition. In competitive inhibition, the K m value increases (the affinity of the enzyme towards substrate is apparently decreased in presence of the inhibitor) whereas V max remains unchanged.

47. Ethanol in the treatment of methanol poisoning Methanol as such is only mildly toxic, in the liver. when acted upon by the enzyme alcohol dehydrogenase, methanol is converted into a highly toxic compound, i.e. formaldehyde. The toxicity of methanol can be overcome by giving ethanol. Ethanol competes with methanol for binding to the active site of the enzyme and slows down the conversion of methanol to formaldehyde, this in turn facilitates the excretion of methanol from the body in the urine, without being converted into formaldehyde.

48. Clinical applications of competitive enzyme inhibition Drug Enzyme True substrate Clinical application Allopurinol Xanthine oxidase Hypoxanthine Gout Sulfonamides Dihydropteroate synthase Para-amino benzoic acid (PABA) Bacterial infection Methotrexate Dihydrofolate reductase Dihydrofolate Cancer Dicumarol Epoxide reductase Vitamin K epoxide Thrombosis Ethanol (alcohol) Alcohol dehydrogenase Ethanol (alcohol) Methanol poisoning Succinyl choline Acetyl cholinesterase Acetyl choline Muscle relaxant Lovastatin , Pravastatin HMG CoA reductase HMG CoA Inhibit cholesterol biosynthesis

49. Sulfonamides Sulfonamides (one of the members of sulfa drugs) has structural similarity to PABA ( para -amino benzoic acid). PABA is required for the biosynthesis of folic acid in bacteria. Sulfanilamide acts as competitive inhibitor of the enzyme, dihydropteroate synthase thus blocking the conversion of PABA to folic acid in bacteria and the bacteria die. Bacterial wall is impermeable to folic acid. The drug is nontoxic to human cells, because human beings cannot synthesize folic acid. Preformed folic acid is essential for man. Sulfonamides are used to treat respiratory and urinary tract infections. Fig. Competitive inhibition

50. Methotrexate It is 4-amino-N 10 – methyl folic acid. It is a structural analog of folic acid, and can competitively inhibit folate reductase enzyme. This is essential for DNA synthesis and cell division. Methotrexate is used as an anticancer drug. Fig. Production of dTMP from dUMP , by the enzyme thymidylate synthase. The reaction needs 1 carbon units and folic acid. DHF = dihydro folic acid. THF = tetrahydro folic acid. Methotrexate inhibits the enzyme DHF-reductase. So dTMP synthesis is inhibited, in turn DNA synthesis is inhibited

51. Dicumarol Due to structural similarity to vitamin K, dicumarol and warfarin act as antagonists to vitamin K. They act as competitive inhibitors of the enzymes concerned with the activation of prothrombin and other coagulation factors which require vitamin K as the coenzyme. Warfarin inhibits the conversion of vitamin K epoxide to vitamin K as well as the conversion of preprothrombin precursor to active prothrombin. They are widely used as anticoagulats for the therapeutic purposes. Fig.Vitamin K cycle. Dicoumarol , a structural analogue inhibits vitamin K reductase

52. Non-competitive inhibition (Irreversible) The inhibitor (I) usually bears no structural similarity to the substrate(S) and thus there occurs no competition between I and S. A non-competitive inhibitor binds at a site other than the substrate binding site hence the enzyme as well as the enzyme-substrate complex can bind to inhibitor. Therefore, both binary(EI) and ternary (ESI) complexes can be formed. Since ESI may breakdown to form a product but at a slower rate, therefore, this type of inhibition is also known as mixed inhibition . Increase in the substrate concentration generally does not relieve this inhibition. The inhibitor combines with the enzymes by forming a covalent bond and then the reaction becomes irreversible. The velocity ( Vmax ) is reduced but Km value is not changed, because the remaining enzyme molecules have the same affinity for the substrate.

53. Fig. Non-competitive inhibition

54. Certain analogs of purines and pyrimidines (called antimetabolites ) are non-competitive inhibitors of some of the enzymes and are used as chemotherapeutic agents, e.g. 5-flurouracil. It is an analog of thymine and inhibits thymidylate synthetase, noncompetitively. Deoxycycline, an antibiotic, functions at low concentrations as a non-competitive inhibitor of a proteolytic enzyme, collagenase . It is used to treat periodontal disease. Metal ions at lower concentrations act as reversible non-competitive inhibitors.

55. Comparison of two types of inhibition Competitive inhibition Non-competitive inhibition Acting on Active site May or may not Structure of inhibitor Substrate analog Unrelated molecule Inhibition is Reversible Irreversible Excess substrate Inhibition relieved No effect Km Increased No change Vmax No change Decreased Significance Drug action Toxicological

56. REGULATION OF ENZYME ACTIVITY IN THE LIVING SYSTEM In biological system, regulation of enzyme activities occurs at different stages in one or more of the following ways. Allosteric regulation Activation of latent enzymes Compartmentation of metabolic pathways Control of enzyme synthesis Enzyme degradation Isoenzymes

57. A llosteric Regulation Allosteric enzymes are regulated by molecules called effectors (also called modifiers) that bind non-covalently at a site other than the active site. The presence of an allosteric effector can alter the affinity of the enzyme for its substrate, or modify the maximal catalytic activity of the enzyme, or both. Effectors that inhibit enzyme activity are termed negative effectors, whereas those that increase enzyme activity are called positive effectors. Allosteric enzymes frequently catalyze the commited step early in a pathway. Feedback inhibition or end product inhibition is a specialized type of allosteric inhibition.

58. Homotropic effectors When the substrate itself serves as an effectors , the effect is said to be homotropic . Most often, an allosteric substrate functions as a positive effector . Positive and negative effectors of allosteric enzymes can affect either the Vmax , the Km, or both. 2. Heterotropic effect The effector may be different from the substrate. For example, the enzyme that converts D to E has an allosteric site that binds the end product, G. If concentration of G increases (for example, because it is not used as rapidly as it is synthesized), the first irreversible step unique to pathway is typically inhibited. The heterotropic effectors are commonly encountered, for example, the glycolytic enzymes phosphofructokinase-1 is allosterically inhibited by citrate, which is not a substrate for the enzyme.

59. Activation of latent enzymes (Covalent modification) Many enzymes may be regulated by covalent modification, most frequently by addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. Phosphorylation and dephosphorylation Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that use ATP as a phosphate donor. Phosphate groups are cleaved from phosphorylated enzymes by the action of phosphoprotein phosphatases . Glycogen phosphorylase is a muscle enzyme that breaks down glycogen to provide energy. This enzyme is a homodimer (two identical subunits) and exists in two interconvertible forms. This enzyme is active in phosphorylated state. There are some enzymes which are active in dephosphorylated state and become inactive when phosphorylated e.g. glycogen synthase, HMG CoA reductase. A few enzymes are active only with sulfhydryl (-SH) groups, e.g. succinate dehydrogenase, urease. Glutathione brings about stability of these enzymes.

60. 2 . Response of enzyme to phosphorylation Depending on the specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme. For example, phosphorylation of glycogen phosphorylase increases activity, whereas the addition of phosphate to glycogen synthase decreases activity. Compartmentation There are certain substances in the body (e.g., fatty acids, glycogen) which are synthesized and also degraded. Generally, the synthetic (anabolic) and breakdown (catabolic) pathways are operative in different cellular organelles to achieve maximum economy. For instance, enzymes for fatty acid synthesis are found in the cytosol whereas enzymes for fatty acid oxidation are present in the mitochondria.

61. Control of enzyme synthesis Most of the enzymes, particularly the rate limiting ones, are present in very low concentration. The amount of the enzyme directly controls the velocity of the reaction, catalysed by the enzyme. Many rate limiting enzymes have short half-lives. This helps in the efficient regulation of the enzyme levels. There are two types of enzymes Constitutive enzymes (house-keeping enzymes) Its levels are not controlled and remain fairly constant. 2. Adaptive enzymes Its concentrations increase or decrease as per body needs and are well regulated.

62. Enzyme degradation Enzymes are not immortal, since it will create a series of problems. There is a lot of variability in the half-lives of individual enzymes. E.g. LDH 4 – 5 to 6 days; LDH 1 – 8 to 12 hours; amylase – 3 to 5 hours. Enzymes when not needed, they immediately disappear and, as when required they are quickly synthesized. Isoenzymes Multiple forms of the same enzyme will also help in the regulation of enzyme activity. Many of the isoenzymes are tissue-specific.

63. APPLICATIONS OF ENZYMES THERAPEUTIC IMPORTANCE Streptokinase : is used in blood clot-dissolution during an acute myocardial infarction or in deep vein thrombosis. Asparaginase : is used for some types of leukemias based on the rationale that the tumor cells are asparagine -dependent for their multiplication and survival. Deoxyribonuclease ( DNAse ) : is administered by the respiratory route to clear up the viscid secretions in patients of cystic fibrosis. 4. Serratiopeptidase : used to minimize the edema that accompanies a physical trauma or an acute inflammation of the skin. 5. Hyaluronidase : Used for hypodermoclysis , to facilitate the subcutaneous administration of water/electrolyte solutions in patients with hypovolemic shock where the collapsed veins are difficult to locate. 6. Hemocoagulase : is used as a hemostat .

64. Some serum enzymes of diagnostic significance Enzyme Principal sources Conditions in which altered ( increase, decrease) Acid phosphatase Prostate, RBC - Carcinoma prostate Alkaline phosphatase Liver, bone, placenta, kidney -Obstructive jaundice, rickets, Paget’s disease, growing children Aldolase Skeletal muscle, brain, heart -Muscular dystrophies, acute hepatitis Amylase Salivary glands -Acute parotitis Amylase Pancreas - Acute pancreatitis, intestinal obstruction Ceruloplasmin Liver -Cirrhosis, Wilson’s disease Creatine phosphokinase Skeletal muscle, brain, heart -Acute myocardial infarction γ - Glutamyl transferase Liver, Kidney -Alcoholism, hepatobiliary disease Glutamate dehydrogenase Liver -Hepatocellular damage

65. Analytical application reagents (for estimation) Enzymes use Glucose oxidase and peroxidase Glucose Urease Urea Cholesterol oxidase Cholesterol Uricase Uric acid Lipase Triacylglycerols Luciferase To detect bacterial contamination of foods Alkaline phosphatase /horse radish peroxidase In the analytical technique ELISA