1. Enzymes Bettelheim, Brown, Campbell and Farrell Chapter 23

2. CO20

3. Enzymes Catalyst: Speeds up rate of reaction but does not change equilibrium. The catalyst itself is not changed. Enzymes are protein molecules which catalyze a chemical reaction

4. Enzymes catalyze specific reactions on specific compounds (single isomers) Business part of enzyme is the “active site” Active site binds the substrate (compound which undergoes a reaction)

5. Enzyme Names Generally come from the name of the reaction that enzyme catalyzes. Frequently end in “ase” Name of substrate - ending + ase Lactose –ose +ase = lactase Alcohol dehydrogenase

6. Categories of Enzymes Oxidoreductases—catalyze redox reaction Transferases—catalyze transfer of functional group to a different molecule –Kinase—transfer of phosphate group –Transaminase—transfer of amino group Hydrolases—hydrolysis reactions (add water and break bond

7. Categories of Enzymes Lyases—add group to double bond OR remove group to make double bond Isomerases—rearrange to make isomer Ligases—join two molecules –May involve several kinds of bonds: C-C C-S C-O C-N

8. How do enzymes catalyze a reaction? Enzymes lower activation energy for a reaction They do not change the equilibrium constant, only the RATE of the reaction

10. Terminology Substrate: compound on which enzyme acts Active site: part of enzyme where substrate binds Activation: process of making an inactive enzyme active Inhibition: process that makes an enzyme less active or inactive –Competitive inhibition –Noncompetitive inhibition

11. Enzyme Activity Enzyme activity:Enzyme activity: a measure of the reaction rate for an enzyme Rate of enzyme-catalyzed reaction is affected by –enzyme concentration –substrate concentration –temperature –pH

12. Enzyme Concentration Enzyme concentration

13. Enzyme Activity

14. Substrate Concentration Rate will level off as sites on enzyme are filled up

15. Initial rate of reaction will double when you double amount of substrate Rate increases to a maximum velocity when all of the active sites on an enzyme are full V max

16. Effect of Temperature Enzyme most active at optimum temperature

17. Effect of Temperature on Enzyme Rate Uncatalyzed reaction rates increase as temperature increases Enzymes have temperature optimum, at which the enzyme has its highest rate –Generally about 37 o C for many enzymes –Above the temperature optimum, enzyme rates fall –At high temperatures, enzyme is denatured

18. Fig. 20.10 Uncatalyzed Reaction Catalyzed Reaction

19. Effect of pH Enzyme most active at optimum pH

20. Effect of pH on Enzyme Rate Enzymes have different reaction rates at different pH’s pH Optimum: pH at which rate is highest –Near pH 7 for many enzymes –Some have optima at very high or low pH At pH higher or lower than optimum, rate falls off At extreme pH, enzyme will be denatured

21. Formation of Enzyme-Substrate Complex E + S ↔ ES ↔ ES* ↔ EP ↔ E + P Enzyme Enzyme Transition EnzymeEnzyme + substrate state product + Substrate complex complex Product Overall Reaction: S → P

22. Enzyme Mechanism Substrate fits into the active site and then undergoes a reaction Enzyme-substrate complex formed Enzyme-substrate complex is an intermediate species

23. Initial binding of substrate relatively fast Conversion of substrate to product (and release of product) is slower This is “Rate Limiting Step”

24. Characteristics of Active Site Site where substrate binds to enzyme Generally have groups that extend into the active site to help catalyze the reaction –Often histidine Substrate “fits” into site. Substrate held by weak, noncovalent interactions in “binding site” Site very specific—only substrate that fits into site will undergo reaction

25. Enzyme Specificity Enzyme specificity is the ability of an enzyme to bind only one (or a very few) substrates and thus catalyze only one reaction

26. Fig. 20.12

27. Levels of Specificity Absolute: One substrate only Group: Similar compounds (hexoses) Linkage: Recognize bond (linkage) types Stereochemical: D- or L- isomer

28. Two Models for Enzyme Activity Lock and Key Model –Emil Fischer 1894 –Substrate fits into “rigid” active site just as a key fits into a lock Induced Fit Model –Daniel Koshland 1958 –Enzyme modifies its shape to accommodate the substrate

29. Induced Fit Model Lock and Key Model

30. –Both the lock-and-key model and the induced-fit model emphasize the shape of the active site –the chemistry of the active site is the most important just five amino acids participate in the active sites in more than 65% of the enzymes studies to date –these five are His > Cys > Asp > Arg > Glu –four of these amino acids have either acidic or basic side chains; the fifth has a sulfhydryl group (-SH)

31. Look at enzyme-substrate complex again Focus on steps in forming transition state and product

32. Formation of Enzyme-Substrate Complex E + S ↔ ES ↔ ES* ↔ EP ↔ E + P Enzyme Enzyme Transition EnzymeEnzyme + substrate state product + Substrate complex complex Product Overall Reaction: S → P

33. How transition state helps reaction to proceed more rapidly Put “stress” on bond in substrate Bring reactants closer together Put reactants into correct orientation Provide different pH environment in active site

34. Stress Bond

35. Correct Orientation

36. Regulation of Enzyme Activity Rate will level off as sites on enzyme are filled up

37. Enzyme Inhibitors Bind to enzymes and eliminate or reduce catalytic activity.

38. Types of Inhibitors Irreversible Inhibitors Reversible, Competitive Inhibitors –Structural Analogs Reversible, Noncompetitive Inhibitors

39. Competitive Inhibition –the induced-fit model explains competitive inhibition –the inhibitor fits into the active site, preventing the substrate from entering

40. Noncompetitive Inhibition Noncompetitive inhibitors –Inhibitor binds elsewhere on enzyme and changes the active site so substrate can’t attach

41. Mechanism of Action –we can distinguish between competitive and noncompetitive inhibition by the enzyme kinetics in the absence and presence of the inhibitor

42. Enzyme Regulation Feedback Inhibition of enzyme pathway A → B → C → D → E → F If enough F is present, it can bind to an enzyme earlier in sequence and inactivate it. This stops synthesis of all subsequent products.

43. Enzyme Regulation Feedback control:Feedback control: an enzyme-regulation process where the product of a series of enzyme- catalyzed reactions inhibits an earlier reaction in a sequence –the inhibition may be competitive or noncompetitive

44. Activating Enzymes Apoenzyme: Protein portion of enzyme Cofactor: Non-protein prosthetic group –Examples: Metal ions such as Zn 2+, Mg 2+ Coenzyme: Organic prosthetic group –Examples: Heme, Vitamins Holoenzyme: Active enzyme

45. Cofactor binds and changes active site

46. Activating enzymes Coenzyme: Bind temporarily to catalytic site to help catalyze reaction (often have vitamin component) Coenzyme binds to apoenzyme first. Substrate binds second Both product and coenzyme are released after reaction

47. 1 2 3 4

48. Examples of Vitamin Coenzymes

49. Regulation of Enzyme Activity Allosteric Enzymes: –More than one binding site –Shape of active site is changed by binding of molecules to another part of enzyme –Regulator molecules bind to regulatory site Positive Allosterism: Changes to active form Negative Allosterism: Changes to inactive form

50. Fig. 20.11

51. Regulation of Enzyme Activity Zymogen (Proenzyme): Enzyme originally made in inactive form. Part of it must be removed before it is active. –Trypsinogen—activated by cleaving off 6 aa Protein Modification: Group can be bound or removed to activate or inactivate an enzyme. Easily reversed. –Phosphorylation of enzyme turns it on/off

52. Examples of Zymogens

53. Isozymes Isozymes (isoenzymes) catalyze the same reaction Different forms in different tissues May be inhibited or turned on by different molecules Frequently have one isozyme active normally Other isozyme can be induced when needed