Understanding Enzymatic Reactions: Thermodynamics and Kinetics

1. BCOR 011 Lecture 12 9/28/2005 ENZYMES

2. Last time… -G reaction “can” go spontaneous But when will it go? And at what rate?

3. Thermodynamics Whether a reaction will occur Kinetics WHEN a reaction will occur

4. What governs WHEN a reaction will occur? The tower of blocks falling is favorable but when will it happen? Oxidation of carbohydrate polymers (starch) to carbon dioxide and water is favorable but when will it happen? Gasoline burning to carbon dioxide and water is favorable but when will it happen ?

5. For a Reaction to occur need to Destabilize Existing State to INPUT ENERGY Destabilization energy input “Activation Energy” Potential net usable energy Potential net usable energy Now In Transition

6. Need to INPUT ENERGY to Destabilize Existing State Regain Activation Energy Invested net usable energy released Potential net usable energy In Transition After

7. What does activation energy represent? For a Reaction to Occur… - reactants must find each other, - meet in proper orientation - and hit with sufficient force

8. Productive Collision Many Non-productive Collisions

9. Needs of Typical chemical reactions - need large number of molecular collisions - need collide violently enough to break pre-existing bonds (not bounce) - need high concentration to find each other at significant rate HEAT !

10. A B D C Transition state B A EA D C Free energy Reactants B A ∆G < O C D Products Progress of the reaction Figure 8.14 The energy profile for an exergonic reaction

11. Molecules with sufficient Energy (~40%) EA Temp 1 Temp 2 Molecules with sufficient Energy (<5%)

12. ENZYMES make reactions easier to occur at reasonable temperature by LOWERING the ACTIVATION ENERGY EA of the reaction

13. Activation Energy “ease” of initiating reaction EA DG Thermodynamic “favorablility” EA DG Energy necessary to overcome the status quo

14. CATALYSTS: promote a specific reaction But are NOTconsumed in the process Keyconcepts: Promotes - does notalter what would normally occur thermodynamically Specificity - promotes only one reaction, only between specific reactants to give specific products Reusable- regenerated in the process

15. ENZYMESare biologicalCATALYSTS - usually PROTEINS - sometimes RNA or RNA/protein complexes

16. Enzymes work as catalysts by providing an easy path to the same point Hard path Easy path HOW?

17. How do Enzymes do it? 1. Enzymes haveBINDING AFFINITY for their reactants =Substrates Brings substrates in close proximity: conc

18. Charged Stabilized Interactions Nonpolar Polar Have a very Specific 3-D Shape With a Specific Arrangement of Functional Groups Flexible OH HO HO + Enzymes act as a Specific Platform

19. ENZYMES: Bind ONLY specific things Bind them ONLY in a Specific 3-D Orientation HO OH OH OH HO - HO + SPECIFICITY is the Key to Enzyme Action

20. 2. Enzymes ORIENT Substrates always in productive orientation

21. Productive Collision ONLY Productive Collisions Many Non-productive Collisions

22. With just a little nudge, can’t help but react HO OH OH OH HO - HO +

23. 3. Enzymes cause BOND STRAIN - destabilize existing bonds “nutcracker effect” 3a. Physical Strain 3b.Chemical Strain

24. Substate Active site Enzyme Figure 8.16 (a) The active site • Is the region on the enzyme where the substrate binds

25. Enzyme- substrate complex (b) Figure 8.16 Induced fit of a substrate

26. Enzyme-substrate interactions Fischer: Lock & key Koshland: Induced fit 3a. Physical bond strain Draw an quarter - an anvil

27. 1 Substrates enter active site; enzyme changes shape so its active site embraces the substrates (induced fit). 2 Substrates held in active site by weak interactions, such as hydrogen bonds and ionic bonds. 3 Active site (and R groups of its amino acids) can lower EA and speed up a reaction by • acting as a template for substrate orientation, • stressing the substrates and stabilizing the transition state, • providing a favorable microenvironment, • participating directly in the catalytic reaction. Substrates Enzyme-substrate complex 6 Active site Is available for two new substrate Mole. Enzyme 5 Products are Released. 4 Substrates are Converted into Products. Figure 8.17 Products • The catalytic cycle of an enzyme

28. 3b. Chemical Bond Strain tease the bond to fall apart

29. Chemical Bond Strain Stabilize a Fictitious state

30. Cofactors Non-polypeptide things at the active site that help enzymes do their job • Cofactors • Are nonprotein enzyme helpers, eg Zn++ • Coenzymes • Are organic cofactors

31. 4. Enzymes “partake” in reactions but are not consumed in them Converts MANY “A’s” into “B’s”

32. H+ OH- H+ Partakes: but start and end with the same enzyme config

33. Lysozyme

34. Lysozyme: kills bacteria Works at pH 4-5 Why?

35. SUMMARY Enzymes: 1. Bring reactants (substrates) in close proximity 2. Align substrates in proper orientation 3. Can act as a Lever: a press or an anvil small shape change translates to large force 4. Release products when reaction done rebind more substrates 5. Many small steps, each easily achieved rather than one huge leap

36. No Problem Dude You expect me to JUMP this? Enzymes carry out reactions in a series of small steps rather than one energetic event

37. Reaction rates: Example: H2O2-> H2O +O2 uncatalyzed –months Fe+++ 30,000x faster Catalase 100,000,000 x faster Enzyme kinetics- kinetikos – moving

38. An enzyme catalyzed rxn Can be “saturated” maximum velocity Rate or velocity 1/2 Vmax # made per min Substrate Conc Km “substrate affinity” Vmax

39. The lower the Km the better the enzyme recognizes substrate “finds it at low conc” “mpg” The higher the Vmax the more substrate an enzyme can process per min (if substrate around) “top speed”

40. Things that affect protein structure often affect enzyme activity temperature 0 20 40 60 80 100 º C pH 0 1 2 3 4 5 6 7 8 9 10 pH

41. Enzyme regulation: Activity controlled Continually adjusted

42. Principal Ways of Regulating Enzymes Competitive Inhibition Allosteric Inhibition Covalent Modification (phosphorylation)

43. S2 S1 Competitive Inhibitors: bind to active site “unproductively” and block true substrates’ access HO OH OH - I HO OH OH HO HO + S & I bind to same site

44. Competitive inhibition

45. Allosteric Inhibitors “other”“site” Distorts the conformation of the enzyme Negative allosteric regulator

46. Allosteric inhibition

47. Positive allosteric regulators Helps enzyme work better promotes/stabilizes an “active” conformation

48. Allosteric activation

49. Allosteric activaterstabilizes active from Allosteric enyzmewith four subunits Active site(one of four) Regulatorysite (oneof four) Activator Active form Stabilized active form Allosteric activaterstabilizes active form Oscillation Non-functionalactivesite Inhibitor Stabilized inactiveform Inactive form (a) Allosteric activators and inhibitors. In the cell, activators and inhibitors dissociate when at low concentrations. The enzyme can then oscillate again. Figure 8.20 Allosteric regulators change the shape conformation of the enzyme

50. A frequent regulatory modification of enzymes Phosphorylation