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Lecture 6

Lecture 6. Enzymology Dr. Nasir Jalal ASAB/NUST. Jokes for your health. Incredible science ScienceDaily (Oct. 2, 2012). Solar Cell Consisting of a Single Molecule: Individual Protein Complex Generates Electric Current.

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Lecture 6

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  1. Lecture 6 Enzymology Dr. NasirJalal ASAB/NUST

  2. Jokes for your health

  3. Incredible scienceScienceDaily (Oct. 2, 2012) Solar Cell Consisting of a Single Molecule: Individual Protein Complex Generates Electric Current The proteins represent light-driven, highly efficient single-molecule electron pumps that can act as current generators in nanoscale electric circuits. The interdisciplinary team publishes the results in Nature Nanotechnology this week. http://www.sciencedaily.com/releases/2012/10/121002150029.htm

  4. Today’s Lecture Article: Particles Walk Through Walls While Physicists WatchClara Moskowitz, LiveScience senior writerDate: 17 May 2012 Time: 07:26 AM ET http://www.livescience.com/20380-particles-quantum-tunneling-timing.html

  5. Electrons can escape from their shell. http://www.livescience.com/20380-particles-quantum-tunneling-timing.html Electrons bound to an atom can sometimes escape, even if they lack the requisite energy, through a phenomenon known as quantum tunneling.CREDIT: Dreamstime

  6. Jokes for your health Why was the turkey playing in the band? Because he had the drumsticks! And then he too was no more.

  7. Electrons can escape from their shell. • Sometimes, particles can pass through walls. • Though it sounds like science fiction, the phenomenon is well documented and even understood under the bizarre rules that govern the microscopic world called quantum mechanics. • Now, scientists have measured the timing of this passing-through-walls trick more accurately than ever before, and report their results in today's (May 17, 2012) issue of the journal Nature. • The process is called quantum tunneling, and occurs when a particle passes through a barrier that it seemingly shouldn't be able to. In this case, scientists measured electrons escaping from atoms without having the necessary energy to do so. In the normal world around us, this would be like a child jumping into the air, and somehow clearing a whole house. http://www.livescience.com/20380-particles-quantum-tunneling-timing.html

  8. Billiards V/S Waves • Quantum tunneling is possible because of the wave-nature of matter. Confounding as it sounds, in the quantum world, particles often act likes waves of water rather than billiard balls. This means that an electron doesn't exist in a single place at a single time and with a single energy, but rather as a wave of probabilities. • Now, physicists led by DrorShafir of Weizmann Institute of Science have prompted electrons to tunnel out of atoms, and measured when they do so to within 200 attoseconds (an attosecond is 10-18 seconds, or 0.000000000000000001 seconds). http://www.livescience.com/20380-particles-quantum-tunneling-timing.html

  9. Quantum tunneling • Quantum tunnelling (or tunneling) is the quantum-mechanical effect of transitioning through a classically-forbidden energy state. • Consider rolling a ball up a hill. • If the ball is not given enough velocity, then it will not roll over the hill. • This makes sense classically. http://www.sciencedaily.com/articles/q/quantum_tunnelling.htm

  10. Quantum tunneling (contd.) • In quantum mechanics, objects do not behave like classical objects, such as balls, do. • On a quantum scale, objects exhibit wavelike behavior. • For a quantum particle moving against a potential hill, the wave function describing the pa. • This wave represents the probability of finding the particle in a certain location, meaning that the particle has the possibility of being detected on the other side of the hill. • This behavior is called tunneling; it is as if the particle has 'dug' through the potential hill. • This means that the particle can extend to the other side of the hill. http://www.sciencedaily.com/articles/q/quantum_tunnelling.htm

  11. Classical mechanics does not explain a particle rolling over the hill. • Normally, the car can only get as far as C, before it falls back again • But a fluctuation in energy could get it over the barrier to E!

  12. Electron wavepacket simulation to explain Quantum tunneling • An electron wavepacket directed at a potential barrier. Note the dim spot on the right that represents tunneling electrons. • This behavior is called tunneling; it is as if the particle has 'dug' through the potential hill.

  13. http://abyss.uoregon.edu/~js/glossary/quantum_tunneling.html

  14. Hydrogen tunnelling in enzyme-catalysed H-transfer reactions: flavoprotein and quinoprotein systems.Sutcliffe MJ, Masgrau L, Roujeinikova A, Johannissen LO, Hothi P, Basran J, Ranaghan KE, Mulholland AJ, Leys D, Scrutton NS.Philos Trans R Soc Lond B Biol Sci. 2006 Aug 29;361(1472):1375-86. • It is now widely accepted that enzyme-catalysed C-H bond breakage occurs by quantum mechanical tunnelling. • This paradigm shift in the conceptual framework for these reactions away from semi-classical transition state theory (TST, i.e. including zero-point energy, but with no tunnelling correction) has been driven over the recent years by experimental studies of the temperature dependence of kinetic isotope effects (KIEs) for these reactions in a range of enzymes, including the tryptophan tryptophylquinone-dependent enzymes such as methylamine dehydrogenase and aromatic amine dehydrogenase, and the flavoenzymes such as morphinonereductase and pentaerythritoltetranitratereductase, which produced observations that are also inconsistent with the simple Bell-correction model of tunnelling. • However, these data especially, the strong temperature dependence of reaction rates and the variable temperature dependence of KIEs-are consistent with other tunnelling models (termed full tunnelling models), in which protein and/or substrate fluctuations generate a configuration compatible with tunnelling.

  15. Just another example of a well • The phenomenon of tunneling, which has no counterpart in classical physics, is an important consequence of quantum mechanics. Consider a particle with energy E in the inner region of a one-dimensional potential well V(x). • (A potential well is a potential that has a lower value in a certain region of space than in the neighbouring regions.) • In classical mechanics, if E < V (the maximum height of the potential barrier), the particle remains in the well forever; • if E > V , the particle escapes. • In quantum mechanics, the situation is not so simple. The particle can escape even if its energy E is below the height of the barrier V , although the probability of escape is small unless E is close to V . In that case, the particle may tunnel through the potential barrier and emerge with the same energy E. http://abyss.uoregon.edu/~js/glossary/quantum_tunneling.html

  16. Jokes for your health Why do scientists park on the street at night, but not in the day? Because they prefer nitrates.

  17. Amino Acids, Proteins, and Enzymes Enzymes Enzyme Action Factors Affecting Enzyme Action Enzyme Inhibition Nomenclature/classification

  18. Enzymes • Catalysts for biological reactions • Most are proteins except ribozymes • Lower the activation energy • Increase the rate of reaction • Activity lost if denatured • May be simple proteins • May contain cofactors such as metal ions or organic (vitamins)

  19. 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

  20. Classification of Enzymes Class Reactions catalyzed • Oxidoreductoases oxidation-reduction • Transferases transfer group of atoms • Hydrolases hydrolysis • Lyases add/remove atoms to/from a double bond • Isomerases rearrange atoms • Ligases combine molecules using ATP

  21. Examples of Classification of Enzymes • Oxidoreductoases oxidases - oxidize ,reductases – reduce • Transferases transaminases – transfer amino groups kinases – transfer phosphate groups • Hydrolases proteases - hydrolyze peptide bonds lipases – hydrolyze lipid ester bonds • Lyases carboxylases – add CO2 hydrolases – add H2O

  22. Examples of enzyme classes Alcohol dehydrogenase/oxidoreductase Glycoside hydolase/hydrolases A–B + H2O → A–OH + B–H PhosphorylasesOr Glutathione S Transferase/transferases ADP+P ATP

  23. Examples of enzyme classes In biochemistry, a lyase is an enzyme that catalyzes the breaking of various chemical bonds by means other than hydrolysis and oxidation, often forming a new double bond or a new ring structure. For example, an enzyme that catalyzed this reaction would be a lyase: ATP → cAMP + PPi Lyases are a class of enzymes that cleave carbon-carbon, carbon-oxygen, phosphorous-oxygen, and carbon-nitrogen bonds by reactions than by hydrolysis or oxidation. This often forms a new double bond or ring structure. For example, the enzyme histidine ammonia-lyase catalyzes the reaction shown below which results in the formation of a double bond. http://www.chem.uwec.edu/Webpapers2005/kasperjm/Pages/page1.html

  24. Isomerase

  25. Ligases Ligase enzymes are enzymes that catalyze reactions which make bonds to join together (ligate) smaller molecules to make larger ones.  Ligase enzymes tend to raise the energy of a system, but the hydrolysis of ATP is often coupled with these reaction to make the reaction spontaneous. DNA ligase catalyzes the joining of the 3′-OH to the 5′-phosphate via a two step mechanism. First, the AMP nucleotide, which is attached to a lysine residue in the enzyme’s active site, is transfered to the 5′-phosphate. Then the AMP-phosphate bond is attacked by the 3′-OH, forming the covalent bond and releasing AMP. To allow the enzyme to carry out further reactions the AMP in the enzyme’s active site must be replenished by ATP. http://www.chem.uwec.edu/Webpapers2005/bloomnl/Pages/Page1.html http://bitesizebio.com/articles/the-basics-how-does-dna-ligation-work/

  26. Learning Check E1 Match the type of reaction with the enzymes: (1) aminase (2) dehydrogenase (3) Isomerase (4) synthetase • Converts a cis-fatty acid to trans. • Removes 2 H atoms to form double bond • Combine two molecules using ATP • Adds NH3

  27. Solution E1 Match the type of reaction with the enzymes: (1) aminase (2) dehydrogenase (3) Isomerase (4) synthetase • 3 Converts a cis-fatty acid to trans. • 2Removes 2 H atoms to form double bond • 4Combine two molecules using ATP • 1Adds NH3

  28. Factors Affecting Enzyme Action: Temperature • Little activity at low temperature • Rate increases with temperature • Most active at optimum temperatures (usually 37°C in humans) • Activity lost with denaturation at high temperatures

  29. Factors Affecting Enzyme Action Optimum temperature Reaction Rate Low High Temperature • A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. • Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results. • Because most animal enzymes rapidly become denatured at temperatures above 40°C, most enzyme determinations are carried out somewhat below that temperature. • Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5°C or below is generally the most suitable. Some enzymes lose their activity when frozen. http://www.worthington-biochem.com/introbiochem/tempeffects.html

  30. Temperature and enzyme activity Amylase enzyme from B. subtiliswas found to be resistant to high temperatures. Enzyme temperatures from 35° to 65° C resulted in very similar enzyme titration curves when the temperature was varied. Interestingly, temperatures very near ambient (25° C) had the greatest reduction in enzyme activity when assessed using the fluorescent amylase substrate.

  31. Factors Affecting Enzyme Action: Substrate Concentration • Increasing substrate concentration increases the rate of reaction (enzyme concentration is constant) • Maximum activity reached when all of enzyme combines with substrate

  32. Factors Affecting Enzyme Action Maximum activity Reaction Rate substrate concentration • The substrate binds to the enzyme through relatively weak forces: • H-bonds • Ionic bonds • van der Waals forces between sterically complementary clusters of atoms. Because rate is no longer dependent on [S] at these high concentrations, the enzyme catalyzed reaction is now obeying zero-order kinetics, i.e., the rate is independent of [S]. This behavior is called the “saturation effect”

  33. Effect of substrate concentration It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum. After this point, increases in substrate concentration will not increase the velocity. This is represented graphically in Figure. Enzyme conc. is limiting the rate of reaction.

  34. Substrate conc. And Vmax It is theorized that when maximum velocity had been reached, all of the available enzyme has been converted to ES, the enzyme substrate complex. This point on the graph is designated Vmax. Using this maximum velocity and equation (given on right), Michaelis developed a set of mathematical expressions to calculate enzyme activity in terms of reaction speed from measurable laboratory data. The Michaelis constant Km is defined as the substrate concentration at 1/2 the maximum velocity. Shown in Figure on previous slide. Using this constant and the fact that Km can also be defined as: Km=K-1 + K2 / K+1 K+1, K-1 and K+2 being the rate constants from equation (above). Michaelis developed the following: Km= [S] Vmax ------------ -1 v

  35. Factors Affecting Enzyme Action: pH • Maximum activity at optimum pH • R groups of amino acids have proper charge • Tertiary structure of enzyme is correct • Narrow range of activity • Most lose activity in low or high pH

  36. Factors Affecting Enzyme Action Optimum pH Reaction Rate 3 5 7 9 11 pH

  37. Effect of pH on enzyme activity Effect of pH on amylase enzyme activity. Amylase enzyme isolated from different organisms were compared for their activity at different pH levels.

  38. A P Gibbs free energy • In a first order chemical reaction, the conversion of A to P occurs because at any given instant: • A fraction of A molecules has the energy necessary to achieve a reactive condition known as the transition state. • In this state, there is a high probability for A to P transition.

  39. Gibbs free energy The rate of any chemical reaction is proportional to the concentration of reactant molecules (A) that have the transition state energy. The height of this energy barrier is called the FREE ENERGY OF ACTIVATION ΔG‡.

  40. Gibbs free energy • Specifically ΔG‡ is the energy required to raise the average energy of 1 mole of reactant (at a given temperature) to the transition-state energy. The relationship between ENERGY OF ACTIVATION ΔG‡ and rate constant k is given by: k=AeΔG‡/RT……………………………..Arrhenius equation A=constant of a particular reaction • Also, 1/k=(1/A)e ΔG‡/RT Take Home message: k is inversely proportional to e ΔG‡/RT . Therefore if the energy of activation decreases, the reaction rate increases.

  41. Learning Check E3 Sucrase has an optimum temperature of 37°C and an optimum pH of 6.2. Determine the effect of the following on its rate of reaction (1) no change (2) increase (3) decrease A. Increasing the concentration of sucrose B. Changing the pH to 4 C. Running the reaction at 70°C

  42. Solution E3 Sucrase has an optimum temperature of 37°C and an optimum pH of 6.2. Determine the effect of the following on its rate of reaction (1) no change (2) increase (3) decrease A. 2, 1 Increasing the concentration of sucrose B. 3 Changing the pH to 4 C. 3 Running the reaction at 70°C

  43. Enzyme Inhibition Inhibitors • Slows down the catalytic activity • Change the protein structure of an enzyme • May be competitive or noncompetitive • Some effects are irreversible

  44. Competitive Inhibition A competitive inhibitor • Has a structure similar to substrate • Occupies active site • Competes with substrate for active site • Has effect reversed by increasing substrate concentration

  45. 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 • No reaction occurs • Effect is not reversed by adding substrate

  46. Learning Check E4 Identify each statement as describing an inhibitor that is (1) Competitive (2) Noncompetitive A. Increasing substrate reverses inhibition • Binds to enzyme, not active site • Structure is similar to substrate D. Inhibition is not reversed with substrate

  47. Solution E4 Identify each statement as describing an inhibitor that is (1) Competitive (2) Noncompetitive A. 1 Increasing substrate reverses inhibition B. 2 Binds to enzyme, not active site C. 1 Structure is similar to substrate D. 2 Inhibition is not reversed with substrate

  48. Cofactors Cofactors and coenzymes : Cofactors and coenzymes Cofactors Some enzymes do not need any additional components to show full activity. However, others require non-protein molecules called cofactors to be bound for activity. Cofactors can be either inorganic ( e.g. , metal ions and iron-sulfur clusters) or organic compounds, (e.g., flavin and heme). Organic cofactors can be either: prosthetic groups, which are tightly bound to an enzyme, or coenzymes, which are released from the enzyme's active site during the reaction. Coenzymes include NADH, NADPH and adenosine triphosphate . These molecules act to transfer chemical groups between enzymes. carbonic anhydrase , with a zinc cofactor bound as part of its active site. These tightly-bound molecules are usually found in the active site and are involved in catalysis. For example, flavin and heme cofactors are often involved in redox reactions. Enzymes that require a cofactor but do not have one bound are called apoenzymes . An apoenzyme together with its cofactor(s) is called a holoenzyme (this is the active form). Most cofactors are not covalently attached to an enzyme, but are very tightly bound. However, organic prosthetic groups can be covalently bound.

  49. Co-enzymes Coenzymes : Coenzymes Coenzymes are small organic molecules that transport chemical groups from one enzyme to another. Some of these chemicals such as riboflavin, thiamine and folic acid are vitamins, (acquired). The chemical groups carried include the hydride ion (H - ) carried by NAD or NADP + , the acetyl group carried by coenzyme A , … etc. Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different enzymes. For example, 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: for example, NADPH is regenerated through the pentose phosphate pathway and S- adenosylmethionine by methionineadenosyltransferase .

  50. Inhibition Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity. activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, chemical environment (e.g. pH),

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