Handout 4

1. Handout 4 Enzymes

2. Enzyme • Catalysts for biological reactions • Most are proteins • Lower the activation energy • Increase the rate of reaction up to 107 fold • Activity lost if denatured • May contain cofactors such as metal ions or organic compounds Catalyst: substance that increase rates of a chemical reaction • does not effect equilibrium • remain unchanged in overall process • reactants bind to catalyst, products are released

3. Chemical Nature of Enzyme Enzymes can be defined as soluble, colloidal, organic catalysts which are produced by living cells but are capable of acting independently of the cells. All enzymes are protein in nature without any exception and exhibit all properties of the proteins. They are water soluble; precipitated by the usual protein precipitating reagents like alcohol, ammonium sulfate and alkaloidal reagents. Extreme alterations of pH and high temperatures denature the enzyme protein and thus make it inactive. All enzymes are globular proteins with a complex three dimensional structure, capable of binding substrate molecules to a part of their surface. Enzymes do not alter the chemical equilibrium point of a reversible reaction but only changed the speed of the reaction.

4. Chemical Nature of Enzyme…. Chemically, the enzymes may be divided into 2 categories: 1. Simple-protein enzymes. These contain simple proteins only e.g., urease, amylase, 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 holoenzymes, e.g., catalase, cytochrome c, etc. Specificity of Enzyme Action. Most enzymes are very specific with respect to the substrates that they act on; i.e., they act either on a single or at the most on some structurally-related substrates. Substrate specificity is often determined by changes in relatively few amino acids in the active site.

5. Classes of Enzyme • Oxidoreductases = catalyze oxidation-reduction reactions (NADH) • Transferases = catalyze transfer of functional groups from one molecule to another. • Hydrolases = catalyze hydrolytic cleavage • Lyases = catalyze removal of a group from or addition of a group to a double bond, or other cleavages involving electron rearrangement. • Isomerases = catalyze intramolecular rearrangement. • Ligases = catalyze reactions in which two molecules are joined. Enzymes named for the substrates and type of reaction.

6. Name of Enzymes • End in –ase • Identifies a reacting substance sucrase – reacts sucrose lipase - reacts lipid • Describes function of enzyme oxidase – catalyzes oxidation hydrolase – catalyzes hydrolysis • Common names of digestion enzymes still use – in pepsin, trypsin

7. Active Site Active site is the region that binds the substrate and converts it into product. It is usually a relatively small part of the whole enzyme molecule and is a 3-dimentional entity formed by amino acid residues that can lie far apart in the linear polypeptide chain. The active site is often a cleft or crevice on the surface of the enzyme that forms a predominantly nonpolar environment which enhances the binding of the substrate. Having bound the substrate molecule, and formed an enzyme-substrate complex, catalytically active residues within the active site of the enzyme act on the substrate molecule to transform it first into the transition state and then into product which is released into solution. The enzyme is now free to bind with another molecule of substrate and begin its catalytic cycle again. The substrate(s) is bound in the active site by multiple weak forces: - electrostatic interactions - hydrogen bonds - van-der Waals bonds - hydrophobic interactions

8. Enzyme Action: Lock and Key Model Two models have been proposed to explain how an enzyme binds its substrate. - lock-and-key model - induced-fit model In the lock-and-key model, the shape of the substrate and the active site of the enzyme are thought to fit together like a key into its lock. The two shapes are considered as rigid and fixed, and perfectly complement each other when brought together in the right alignment. • Only certain substrates can fit the active site • Amino acid R groups in the active site help substrate bind • Enzyme-substrate complex forms • Substrate reacts to form product • Product is released.

9. Lock and Key Model + + E + S ES complex E + P P S S P

10. Enzyme Action: Induced Fit Model In the induced-fit model, the binding of substrate induced a conformational change in the active site of the enzyme. In addition, the enzyme may distort the substrate, forcing it into a conformation similar to that of the transition state. • Enzyme structure flexible, not rigid • Enzyme and active site adjust shape to bind substrate • Increases range of substrate specificity • Shape changes also improve catalysis during reaction • For example, the binding of glucose to hexokinase induces a conformational change in the structure of the enzyme such that the active site assumes a shape that is complementary to the substrate (glucose) only after it has bound to the enzyme.

11. Enzyme Action: Induced Fit Model • E + S ES complex E + P

12. Enzymatic Catalysis The energy changes that take place during the course of a particular biochemical reaction (Figure). Activation Energy and Transition State In all reactions there is an energy barrier that has to be overcome in order for the reaction to proceed. That is the energy needed to transform the substrate molecules into the transition state – an unstable chemical part-way between the substrates and the products. The transition state (X#) has the highest free energy of any component in the reaction pathway. The Gibb free energy of activation (ΔG#) is equal to the differences in free energy between the transition state and the substract (Fig.).

13. Enzymatic Catalysis…. Activation Energy and Transition State An enzyme work by stabilizing the transition state of a reaction and decreasing ΔG#. The enzyme does not alter the energy levels of the substrates or the products. Thus an enzyme increases the rate at which the reaction occurs but has no effect on the overall change in energy of the reaction.

14. Enzymatic Catalysis….. Enzymatic Catalysis • Activation Energy (AE) – The energy require to reach transition state from ground state. • AE barrier must be exceeded for reaction to proceed. • Lower AE barrier, the more stable the transition state (TS) • The higher [TS], the move likely the reaction will proceed.

15. Enzyme binding of substrates decrease activation energy by increasing the initial ground state (brings reactants into correct orientation, decrease entropy) • Need to stabilize TS to lower activation energy barrier.

16. k1 k2 E + S ES E + P k-1 k-2 Enzyme Kinetics Michaelis-Menten Model uses the following concept of enzyme catalysis: E = Enzyme S = Substrate P = Product ES = Enzyme-Substrate complex Where the rate constants k1, k-1 and k2 describe the rates associated with each step of the catalytic process. k1 rate constant for the forward reaction k-1 = rate constant for the breakdown of the ES to substrate k2 = rate constant for the formation of the products. It is assumed that there is no significant rate for the backward reaction of enzyme and product (E + P) being converted to ES complex

17. Kinetics of Enzymes… From the observation of the properties of many enzymes it was known that the initial velocity (Vo) at low substrate concentration is directly proportional to [S], while at high substrate concentration the velocity tends towards a maximum value, that is the rate becomes independent of [S]. This maximum velocity is called Vmax. Michaelis-Menten derived an equation to describe these observations, the Michaelis-Menten equation: Where, Vo = velocity at a given concentration of substrate Vmax = maximal velocity possible with excess of [s] [S] = concentration of substrate at velocity Vo Km = Michaelis constant for enzyme. A mathematically evolved relationship between the substrate concentration and velocity of reaction.

18. Kinetics of Enzymes…. Michaelis-Menten equation describes a hyperbolic curve Plot Vo vs. [S]

19. k2 k1 E + S ES E + P k-1 k-2 Kinetics of Enzymes…. In deriving the equation, Michaelis-Menten defined a new constant, Km, the Michaelis constant. Km is a measure of the stability of the ES complex, being equal to the sum of the rates of breakdown of ES over its rate of formation. For many enzymes k2is much greater than k-2. Under these circumstances Km becomes a measure of the affinity of an enzyme for its substrate since its value depends on the relative values of k1and k2 for ES formation and dissociation, respectively. A high Km indicates weak substrate binding (k2 predominant over k1 ), a low Km indicates strong substrate binding.

20. Kinetics of Enzymes…. Km can be determined experimentally by determining the reaction rates i.e., the velocity of reaction (relative activity of enzyme) at various substrate concentration, when Vo= ½ Vmax . By the fact that its value is equivalent to the substrate concentration at which the velocity is equal to half of Vmax . The Km is the substrate concentration where vo equals one-half Vmax

21. Kinetics of Enzymes… It is possible to choose [S] as to make Vo= ½ Vmax . Then ½ Vmax = Dividing by Vmax then ½ = [S]/Km + [S] Therefore, Km + [S] = 2 [S] and Km = [S] Km is independent of enzyme concentration. Hence, Km, the Michaelis constant, can be defined as the conc. of the substrate when the velocity of the enzyme reaction is half their maximal possible. The Km value varies from enzyme to enzyme and is used in characterizing the different enzyme. Most enzyme. Km value varies between 10-1 and 10-6M. Vmax = velocity where all of the enzyme is bound to substrate (enzyme is saturated with S) • Vo = Vmax [S] • Km + [S] Vmax [S] Km + [S] Km = [S] @ ½ Vmax (units moles/L=M) (1/2 of enzyme bound to S)

22. What does Km mean? • Km = [S] at ½ Vmax • Km represents the amount of substrate required to bind ½ of the available enzyme (binding constant of the enzyme for substrate) • Km can be used to evaluate the specificity of an enzyme for a substrate (if obeys M-M) • Small Km means tight binding; high Km means weak binding Glucose Km = 8 X 10-6 Allose Km = 8 X 10-3 Mannose Km = 5 X 10-6 Hexose Kinase Glucose + ATP <-> Glucose-6-P + ADP

23. Kinetics of Enzymes… The method describe above for the determination of Km is somewhat complex and therefore, simpler methods have been devised. Because Vmax is achieved at infinite substrate concentration. It is impossible to estimate Vmax (and hence Km) from a hyperbolic plot. A convenient means of evaluating Km and Vmax is to plot kinetic data as the reciprocals of Vo and [S]. If one takes the reciprocals of Michaelis-Menten equation: The following equation is obtained: This is known as Lineweaver-Burk equation . This equation is of the form y= mx + b, a straight line. If one considered the variables to be 1/Vo and 1/[S]. When ploted, a straight line is obtained.

24. Lineweaver-Burke Plots(double reciprocal plots) slope = KM/Vmax, 1/vo intercept is equal to 1/Vmax the extrapolated x intercept is equal to -1/KM Since Vmax can be determined from the intercept, the Km may also be calculated. • Plot 1/[S] vs 1/Vo • L-B equation for straight line

25. Factors Affecting Enzyme Activity • Contact between the enzyme and substrate • The enzyme being a protein forming a colloidal solution, the substrate also must be a water soluble substance. • If the substrate is a lipid it must be emulsified to enable it to come into contact with the enzyme. • Thus pancreatic lipase can act on lipids in the gastrointestinal tract only when the lipid is emulsified by bile salts. • Substrate Concentration • Increasing substrate concentration increases the rate of reaction (enzyme concentration is constant) • Maximum activity reached when all of enzyme combines with substrate. Further increase in substrate concentration does not increase rate of reaction.

26. Factors Affecting Enzyme Activity: Substrate Concentration Maximum activity Reaction Rate Substrate concentration 

27. Factors Affecting Enzyme Activity… • Enzyme Concentration • Within reasonable limits, the rate of an enzyme reaction steadily increases with increasing concentration of the enzyme • The relationship is linear, showing a direct proportionality. • pH • Maximum activity at optimum pH • The optimal pH for pepsin is around 2.0, while that of trypsin varies from pH 8.0 to 9.0. • Most lose activity in low or high pH

28. Factors Affecting Enzyme Activity: pH Reaction Rate Optimum 3 5 7 9 11 pH

29. Factors Affecting Enzyme Activity • Concentration of the Products • Accumulation of products of reaction causes a lowering of the enzyme activity. • This is prevented in nature by prompt removal of products from the site of formation • Temperature • Little activity at low temperature (inactive at 0°C ) • Rate increases with temperature • Most active at optimum temperatures (usually 37°C in humans) • Activity lost with denaturation at high temperatures (at 70 to 80°C)

30. Factors Affecting Enzyme Activity: Temperature Enzymes can be stored for years by keeping in a frozen state. They regain full activity if they are brought back to laboratory temperature. Optimum temperature Reaction Rate Low High Temperature The temperature at which an enzyme shows maximum activity is known as the optimum temperature for the enzyme.

31. Factors Affecting Enzyme Action: • Effect of Oxidizing Substances • The activity of many enzymes, particular the hydrogenases depends on –SH groups. • The activity is lost if the –SH groups are oxidized to –S-S- groups. • Reducing substances like glutathione and cysteine can reactivate such enzymes. • Radiation • Exposure to UV rays, X-rays, β- and γ-rays causes the formation of peroxides which oxidize the enzymes and make them inactive.

32. Enzyme Inhibitor / Inhibition • Inhibitor – substance that binds to an enzyme and interferes with its activity • Can prevent the formation of ES complex or prevent ES breakdown to E + P. • Irreversible and Reversible Inhibitors • Irreversible inhibitor binds to enzyme through covalent bonds (binds irreversibly) • Reversible Inhibitors bind through non-covalent interactions (disassociates from enzyme) • Inhibition cause a loss of catalytic activity • Inhibition change the protein structure of an enzyme.

33. Enzyme Inhibition… • Enzyme inhibition may be of two main types: irreversible or reversible, depending on whether the enzyme-inhibitor (EI) complex dissociates rapidly or very slowly. • A reversible inhibitor dissociates very rapidly from its target enzyme because it becomes very loosely bound with the enzyme. Three general types of reversible inhibition are distinguished competitive, noncompetitive and uncompetitive,depending on three factors: • Whether the inhibition is or is not overcome by increasing the concentration of the substrate, • Whether the inhibitor binds at the active site or at allosteric site, and • Whether the inhibitor binds either with the free enzyme only, or with the enzyme substrate (ES) complex only or with either of the two.

34. Irreversible Inhibition Irreversible inhibitors are those that combine with or destroy a functional group on the enzyme that is essential for its activity. An irreversible inhibitor binds tightly, often covalently, to amino acid residues at the active site of the enzyme permanently inactivating the enzyme. Example: diisopropylphosphofluoridate (DIPF), a component of nerve gases, reacts with a Ser residue in the active site of the enzyme acetylcholinresterase, an enzyme critical for the transmission of nerve impulses.

35. Irreversible Inhibition DIPF = DIFP = diisopropylfluorophosphate

36. Competitive Inhibition A competitive inhibitor • Has a structure similar to substrate • Occupies active site • Competes with substrate for active site (for enzyme binding) • Can binds with free enzyme forming an enzyme inhibitor (EI) complex rather than an ES complex • The inhibitor binds reversibly to the active site • At high substrate concentration can relieve inhibition Enzyme-inhibitor complex does not yield any products and hence remains stable, thus preventing further enzyme activity. The degree of inhibition depends upon the relative concentration of the substrate and the inhibitor. Thus can be reversed by adding excess of substrate which will successfully dislodge the inhibitor molecules from the enzyme

37. Inhibition kinetics…. Competitive- Where the inhibitor competes with the substrate. A competitive inhibitor increases the slope of the line on the Lineweaver-Burke plot, and alters the intercept on the x-axis (since Km is increased), but leaves the intercept on the y-axis unchanged (since Vmax remain constant). No change in Vmax of the enzyme but the apparent affinity of the enzyme for its substrate decreases in the presence of the competitive inhibitor, and hence Km increases, since -1/Km is decreases. In competitive inhibition, the enzyme can bind substrate (forming an ES complex) or inhibitor (EI), but not both substrate and inhibitor (ESI). Competitive Inhibition: Lineweaver-Burke Plot

38. C-OO- C-H C-H C-OO- C-OO- H-C-H H-C-H C-OO- C-OO- H-C-H H-C-H H-C-H C-OO- C-OO- H-C-H C-OO- C-OO- C-OO- Competitive Inhibition Substrate Product Competitive Inhibitor Fumarate Succinate Glutarate Malonate Oxalate Succinate Dehydrogenase Succinate dehydrogenase can be inhibited by malonate, oxalate and glutarate, all of which resemble the structure of succinic acid, the substrate of the enzyme.

39. Noncompetitive Inhibition A noncompetitive inhibitor • Does not have a structure like substrate • Binds to the enzyme but not active site • Changes the shape of enzyme and active site • Substrate cannot fit altered active site • Effect is not reversed by adding substrate • Can bind free E or ES complex Since [I] and [S] may combine at different sites, simultaneous formation of both [EI] and [ESI] complexes can takes place. Both [ES] and [ESI] may break down to produce the reaction product [P]. In contrast to competitive inhibition, inhibition cannot be overcome by increasing substrate concentration. The enzyme is inactivated when inhibition is bound, whether or not substrate is also present. Thus, a noncompetitive inhibitor acts by lowering the turnover number rather than by decreasing the proportion of enzyme molecules that are bound to the substrate.

40. Inhibition kinetics…. In non-competitive inhibitor the affinity of the enzyme for the substrate is unchanged and so Km remains the same. A L-B plot, it increases the slope and alters the intercept on the y-axis (since Vmax is decreased), but leaves the intercept on the x-axis unchanged (since Km remains constant). The inhibitor effectively lowers the concentration of active enzyme and hence lowers Vmax ( 1/Vmax is increased), but does not alter the Km. • Lowers Vmax, but Km remains the • same • NI’s don’t bind to S binding site • therefore don’t effect Km • Alters conformation of enzyme to • effect catalysis but not substrate • binding Non-competitive Inhibition: Lineweaver-Burke Plot

41. Examples of Noncompetitive Inhibition Various heavy metal ions (Ag+, Hg2+, Pb2+) 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 enzyme: Enz –SH + Ag+ Enz–S–Ag + H+ Because of the reversibility of mercaptide formation, the inhibition can be relieved by removal of the heavy metal ion. In the treatment of lead poisoning, advantage is taken of the metal’s affinity for –SH groups. Therefore, the sulfhydral compounds are administered to interact with the metal in the circulatory system and form mercaptides, which are then excreted. Similarly, cyanide and H2S strongly inhibit the action of iron-containing enzymes like catalase and peroxidase.

42. Uncompetitive Inhibition When the inhibitor combines with the ES complex instead of E and causes inhibition, it is sometimes called uncompetitive inhibition. The ESI complex can not form any products. Prevents ES from proceeding to E + P or back to E + S. • Lowers Km and Vmax, but ratio of Km/Vmax remains the same. • The inhibition is not reversed by increasing substrate concentration. • Occurs with multisubstrate enzymes. Uncompetitive inhibition is much less common in nature. Lithium, a drug used in mania is a good example, it inhibit inositol monophosphatase (IMPase) and prevent recycling of inositol for signaling pathway.

44. Cofactors, Coenzymes and Prosthetic Groups Some enzymes require the presence of cofactors, small non-protein units, to function. Cofactors may be inorganic ions (Zn++ or Fe++) or a complex organic molecule called coenzyme. Coenzymes • Non-protein molecules that help enzymes function • Associate with active site of enzyme • Enzyme + Co-enzyme = holoenzyme • Enzyme alone = apoenzyme • Organic cofactor : co-enzymes – thiamin, riboflavin, niacin, biotin • Inorganic cofactors: essential ions – Mg ++, Fe++, Zn++, Mn++ The coenzymes seem to function by helping in the carriage or transport of one of the products of the enzyme reaction. A metal or coenzyme that is covalently attached to the enzyme is called a prosthetic group (heme in hemoglobin).

45. Digestive Enzymes Our food is made up of: • Carbohydrates: Bread, Pasta, Potato • Protein: Meat, Nuts, Lentils • Fats: Butter, Milk Our body needs to digest them – turn them into a form that can be absorbed into the blood and used by cells. Enzymes make this possible. Enzymes help in the breakdown of food, in a process called digestion. Food contains carbohydrates, proteins and lipids, so a wide range of enzymes is needed.

46. Digestive Enzymes: Carbohydrate Digestion Carbohydrate digestion involves two stages: • First the breakdown of starch to maltose is catalysed by the enzyme amylase in the mouth and the lumen of the small intestine. • Secondly the breakdown of maltose to glucose is catalysed by the enzyme maltase inside the mucosa cells of the small intestine.

47. Digestive Enzymes: Protein Digestion • Protein digestion in the lumen of the gut starts with an enzyme called endopeptidase that catalyses the breakdown of proteins to form polypeptides. • An enzyme called an exopeptidase catalyses the breakdown of polypeptides to produce dipeptides. • Inside the cells of the mucosa dipeptidase enzymes catalyse the breakdown of dipeptides into amino acids.

48. Digestive Enzymes: Lipid Digestion • Lipid digestion only occurs in the lumen of the small intestine. • Lipid digestion cannot start in the stomach because conditions are too acidic for the lipase enzymes. • Bile salts found in bile produced by the liver break down the fat droplets into smaller droplets. • This process is called emulsification. It increases the surface area for the lipase enzymes to work on. • Lipase from the pancreas catalyses the breakdown of lipids into fatty acids and glycerol.