1. HOW ENZYMES WORK

2. ENZYMES SPEED UP CHEMICAL REACTIONS Enzymes are biological catalysts – substances that speed a reaction without being altered in the reaction. Most enzymes are proteins. Enzymes are essential for life. Model of the surface of an enzyme.

3.  Enzymes Cofactors Coenzymes Holoenzyme Apoenzyme

4. Body conditions(temperature, pressure etc.) not good for reaction Only enzymes can catalyse the reactions in this conditions A special environment inside enzymes for reaction ACTIVE SITE Molecule binds active site SUBSTRATE How Enzymes Work?

8. Enzymes Lower a Reaction’s Activation Energy

9. Each reaction has a transition state where the substrate is in an unstable, short-lived chemical/structural state. Free Energy of Activation is symbolized by ΔG ‡. Enzymes act by lowering the free energy of the transition state

10. Enzymes speed up metabolic reactions by lowering energy barriers Enzyme speed reactions by lowering EA. – The transition state can be reached at moderate temperatures. Enzymes do not change delta G. – It speed-up reactions that would occur eventually. Because enzymes are so selective, they determine which chemical processes will occur at any time

11. Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate The enzyme must bind the substrate in the correct orientation otherwise there would be no reaction Not a lock & key but induced fit – the enzyme and/or the substrate distort towards the transition state

13. Induced Fit shape A change in the shape of an enzyme’s active site Induced Induced by the substrate

14. Lock and Key Model An enzyme binds a substrate in a region called the active site 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

16. Enzyme Kinetics - Kinetics The study of the rate of change. - Enzyme Kinetics Rate of chemical reactions mediated by enzymes. Enzymes can increase reaction rate by favoring or enabling a different reaction pathway with a lower activation energy, making it easier for the reaction to occur.

17. Michaelis-Menten kinetics V 0 varies with [S] V max approached asymptotically V 0 is moles of product formed per sec. when [P] is low (close to zero time) E + S  ES  E + P Michaelis-Menten Model V 0 = V max x[S]/([S] + K m ) Michaelis-Menten Equation

18. Determining initial velocity (when [P] is low)

19. Steady-state & pre-steady-state conditions At equilibrium, no net change of [S] & [P] or of [ES] & [E] At pre-steady-state, [P] is low (close to zero time), hence, V 0 for initial reaction velocity At pre-steady state, we can ignore the back reactions

20. Michaelis-Menten kinetics (summary) Enzyme kinetics (Michaelis-Menten Graph) : At fixed concentration of enzyme, V0 is almost linearly proportional to [S] when [S] is small, but is nearly independent of [S] when [S] is large Proposed Model: E + S  ES  E + P ES complex is a necessary intermediate Objective: find an expression that relates rate of catalysis to the concentrations of S & E, and the rates of individual steps Start with: V 0 = k 2 [ES], and derive, V 0 = V max x[S]/([S] + K m ) This equation accounts for graph data. At low [S] ([S] < K m ), V 0 = (V max /K m )[S] At high [S] ([S] > K m ), V 0 = V max When [S] = K m, V 0 = V max /2. Thus, K m = substrate concentration at which the reaction rate (V 0 ) is half max.

21. Range of K m values K m provides approximation of [S] in vivo for many enzymes

22. Lineweaver-Burk plot (double-reciprocal)

23. Eadie-Hofstee plot

24. Hanes-Woolf Plot

25. Allosteric Enzymes Allosteric enzymes have one or more allosteric sites Allosteric sites are binding sites distinct from an enzyme’s active site or substrate-binding site Molecules that bind to allosteric sites are called effectors or modulators Binding to allosteric sites alters the activity of the enzyme. This is called cooperative binding. Allosteric enzymes display sigmoidal plot of Vo vs [S] Effectors may be positive or negative Effectors may be homotropic or heterotropic Regulatory enzymes of metabolic pathways are allosteric enzymes (eg: feedback inhibition)

26. Allosteric enzymes  Allosteric enzymes tend to be multi-sub unit proteins  The reversible binding of an allosteric modulator (here a positive modulator M) affects the substrate binding site

28. [S] vovo Mechanism and Example of Allosteric Effect A I [S] vovo vovo (+) (-) X X X R = Relax (active) T = Tense (inactive) Allosteric site Homotropic (+) Concerted Heterotropic (+) Sequential Heterotropic (-) Concerted Allosteric site KineticsCooperationModels (-) (+)

29. Enzyme Inhibitors Specific enzyme inhibitors regulate enzyme activity and help us understand mechanism of enzyme action. (Denaturing agents are not inhibitors) Irreversible inhibitors form covalent or very tight permanent bonds with aa at the active site of the enzyme and render it inactive. 3 classes: groupspecific reagents, substrate analogs, suicide inhibitors Reversible inhibitors form an EI complex that can be dissociated back to enzyme and free inhibitor. 3 groups based on their mechanism of action: competitive, non-competitive and uncompetitive.

30. Enzyme Inhibition

31. Competitive inhibitors Compete with substrate for binding to enzyme E + S = ES or E + I = EI. Both S and I cannot bind enzyme at the same time In presence of I, the equilibrium of E + S = ES is shifted to the left causing dissociation of ES. This can be reversed / corrected by increasing [S] Vmax is not changed, KM is increased by (1 + I/Ki) Eg: AZT, antibacterial sulfonamides, the anticancer agent methotrexate etc

32. Competitive Inhibition

33. Kinetics of competitive inhibitor Increase [S] to overcome inhibition V max attainable, K m is increased K i = dissociation constant for inhibitor

34. V max unaltered, K m increased

35. Non-competitive Inhibitors Inhibitor binding site is distinct from substrate binding site. Can bind to free enzyme E and to ES E + I = EI, ES + I = ESI or EI + S = ESI Both EI and ESI are enzymatically inactive The effective functional [E] (and [S]) is reduced Reaction of unaffected ES proceeds normally Inhibition cannot be reversed by increasing [S] KM is not changed, Vmax is decreased by (1 + I/Ki)

36. Mixed (Noncompetitive) Inhibition

37. Kinetics of non-competitive inhibitor Increasing [S] cannot overcome inhibition Less E available, V max is lower, K m remains the same for available E

38. K m unaltered, V max decreased

39. Uncompetitive Inhibitors The inhibitor cannot bind to the enzyme directly, but can only bind to the enzyme-substrate complex. ES + I = ESI Both Vmax and KM are decreased by (1+I/Ki).

40. Uncompetitive Inhibition

41. Substrate Inhibition Caused by high substrate concentrations E + S ES E + P Km’Km’ k2k2 K S1 + S ES 2

42. Substrate Inhibition At low substrate concentrations [S] 2 /K s1 <<1 and inhibition is not observed Plot of 1/v vs. 1/[S] gives a line Slope = K ’ m /V m Intercept = 1/V m

43. Substrate Inhibition At high substrate concentrations, K ’ m /[S]<<1, and inhibition is dominant Plot of 1/v vs. [S] gives a straight line Slope = 1/K S1 · V m Intercept = 1/V m

44. 1/V I>0 I=0 1/V m -1/K m -1/K m,app 1/[S] 1/V I>0 I=0 1/V m -1/K m -1/K m,app 1/[S] 1/V m,app 1/V I>0 I=0 1/V m -1/K m 1/[S] 1/V m,app 1/V 1/V m -1/K m 1/[S] Competitive Uncompetitive Non-Competitive Substrate Inhibition

45. Enzyme Inhibition (Mechanism) Competitive Non-competitive Uncompetitive E E Different site Compete for active site Inhibitor Substrate Cartoon Guide Equation and Description I [ I ] binds to free [E] only, and competes with [S]; increasing [S] overcomes I Inhibition by [ I ]. I [ I ] binds to free [E] or [ES] complex; Increasing [S] can I not overcome [ I ] inhibition. I [ I ] binds to [ES] complex only, increasing [S] favors I the inhibition by [ I ]. E + S → ES → E + P + I ↓ I E I ← ↑ E + S → ES → E + P + + II I I ↓ II E I + S →E I S ← ↑ ↑ E + S → ES → E + P + I ↓ I E I S ← ↑ X

46. KmKm Enzyme Inhibition (Plots) CompetitiveNon-competitive Uncompetitive Direct Plots Double Reciprocal V max KmKm Km’Km’[S], mM vovo vovo II K m [S], mM V max I Km’Km’ V max ’ V max unchanged K m increased V max decreased K m unchanged Both V max & K m decreased I 1/[S]1/K m 1/v o 1/ V max I Two parallel lines I Intersect at X axis 1/v o 1/ V max 1/[S]1/K m 1/[S]1/K m 1/ V max 1/v o Intersect at Y axis = Km’= Km’

47. Factors Affecting Enzyme Kinetics

48. Effects of pH - on enzymes - enzymes have ionic groups on their active sites. - Variation of pH changes the ionic form of the active sites. - pH changes the three-Dimensional structure of enzymes. - on substrate - some substrates contain ionic groups - pH affects the ionic form of substrate affects the affinity of the substrate to the enzyme.

49. Effects of Temperature Reaction rate increases with temperature up to a limit Above a certain temperature, activity decreases with temperature due to denaturation Denaturation is much faster than activation Rate varies according to the Arrhenius equation Where E a is the activation energy (kcal/mol) [E] is active enzyme concentration

50. Factors Affecting Enzyme Kinetics  Temperature - on the rate of enzyme catalyzed reaction k 2 =A*exp(-Ea/R*T) T k 2 - enzyme denaturation T Denaturation rate: k d =A d *exp (-Ea/R*T) k d : enzyme denaturation rate constant; E a : deactivation energy

51. REFERENCES Michael L. Shuler and Fikret Kargı, Bioprocess Engineering: Basic Concepts (2 nd Edition),Prentice Hall, New York, 2002. 1. James E. Bailey and David F. Ollis, Biochemical Engineering Fundementals (2 nd Edition), McGraw-Hill, New York, 1986. www.biochem.umass.edu/courses/420/le ctures/Ch08B.ppt -

52. class.fst.ohio- state.edu/fst605/605p/Enzymes.pdf – www.horton.ednet.ns.ca/staff/selig/powerpoint s/bio12/biochem/enzymes.pdf www.siu.edu/departments/biochem/som_pbl/S SB/powerpoint/enzymes.ppt www.associazioneasia.it/adon/files/2005_luisi_ 05_why_are_enzymes.pdf www.fatih.edu.tr/~abasiyanik/Chapter6_enzym es.pdf -

53. http://www.authorstream.com/presentation/kkoza r-14001-enzymes-enzyme-ppt-education- powerpoint/ http://highered.mcgraw- hill.com/sites/0072495855/student_view0/chapte r2/animation__how_enzymes_work.html http://www.wiley.com/college/pratt/0471393878/s tudent/animations/enzyme_kinetics/index.html