1. ENZYMES A protein with catalytic properties due to its power of specific activation IB Topics 3.6 & 7.6 Material on this power point adapted from Paul Billiet ODWSODWS

2. Chemical reactions  Chemical reactions need an initial input of energy = THE ACTIVATION ENERGY  During this part of the reaction the molecules are said to be in a transition state.

3. Reaction pathway

4. How Enzymes Affect Reaction Rates Enzymes affect the rates of reactions by lowering the amount of energy of activation required for the reactions to begin. Therefore processes can occur in living systems at lower temperatures or energy levels than it would require for these same reactions to occur without the enzymes present.

5. How Enzymes Affect Reaction Rates C 6 H 12 0 6 + 60 2  6CO 2 + 6H 2 0 + ENERGY Above is the formula for the complete combustion of glucose. This reaction can be carried out in a laboratory at several hundred degrees Celsius. The same reaction occurs during the process of cellular respiration in living cells. In humans at a temperature of 37 o Celsius. What makes the difference? ENZYMES!

6. Enzyme structure  Enzymes are proteins  They have a globular shape  A complex 3-D structure Human pancreatic amylase © Dr. Anjuman BegumDr. Anjuman Begum

7. The active site  One part of an enzyme, the active site, is particularly important  The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily © H.PELLETIER, M.R.SAWAYA ProNuC Database ProNuC Database

8. Cofactors  An additional non- protein molecule that is needed by some enzymes to help the reaction  Tightly bound cofactors are called prosthetic groups  Cofactors that are bound and released easily are called coenzymes  Many vitamins are coenzymes Nitrogenase enzyme with Fe, Mo and ADP cofactors Jmol from a RCSB PDB file © 2007 Steve Cook H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES2007 Steve Cook STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

9. The substrate  The substrate of an enzyme are the reactants that are activated by the enzyme  Enzymes are specific to their substrates  The specificity is determined by the active site

10. How Enzymes Bind to Substrates  There are two proposed methods by which enzymes bind to their substrate molecules: a. Lock and Key Model b. Induced-Fit Model

11. The Lock and Key Hypothesis  Fit between the substrate and the active site of the enzyme is exact  Like a key fits into a lock very precisely  The key is analogous to the enzyme and the substrate analogous to the lock.  Temporary structure called the enzyme-substrate complex formed  Products have a different shape from the substrate  Once formed, they are released from the active site  Leaving it free to become attached to another substrate

12. The Lock and Key Hypothesis Enzyme may be used again Enzyme- substrate complex E S P E E P Reaction coordinate

13. The Lock and Key Hypothesis  This explains enzyme specificity  This explains the loss of activity when enzymes denature

14. The Induced Fit Hypothesis  Some proteins can change their shape (conformation)  When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation  The active site is then moulded into a precise conformation  Making the chemical environment suitable for the reaction  The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

15. The Induced Fit Hypothesis  This explains the enzymes that can react with a range of substrates of similar types Hexokinase (a) without (b) with glucose substrate http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

16. Factors affecting Enzymes  substrate concentration  pH  temperature  inhibitors

17. Substrate concentration: Non-enzymic reactions  The increase in velocity is proportional to the substrate concentration Reaction velocity Substrate concentration © 2007 Paul Billiet ODWSODWS

18. Substrate concentration: Enzymic reactions  Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied.  If you alter the concentration of the enzyme then V max will change too. Reaction velocity Substrate concentration V max

19. The effect of pH  Extreme pH levels will produce denaturation (the structure of the enzyme is changed)  The active site is distorted and the substrate molecules will no longer fit in it  At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur  This change in ionization will affect the binding of the substrate with the active site.

20. The effect of pH Optimum pH values Enzyme activity Trypsin Pepsin pH 1 3 5 7 9 11

21. The effect of temperature  Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in temperature.  For chemical reactions the Q10 = 2 to 3 (the rate of the reaction doubles or triples with every 10°C rise in temperature)  Enzyme-controlled reactions follow this rule as they are chemical reactions  BUT at high temperatures proteins denature  The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation.

22. The effect of temperature Temperature / °C Enzyme activity 0 10 2030 4050 Q10 Denaturation

23. The effect of temperature  For most enzymes the optimum temperature is about 30°C  Many are a lot lower, cold water fish will die at 30°C because their enzymes denature  A few bacteria have enzymes that can withstand very high temperatures up to 100°C  Most enzymes however are fully denatured at 70°C

24. Inhibitors  Inhibitors are chemicals that reduce the rate of enzymic reactions.  The are usually specific and they work at low concentrations.  They block the enzyme but they do not usually destroy it.  Many drugs and poisons are inhibitors of enzymes in the nervous system.

25. The effect of enzyme inhibition There are two forms of inhibition: 1. Competitive inhibition 2. Noncompetitive inhibition

26. Competitive Inhibition This type of inhibition occurs when another molecule is structurally similar to the substrate and can bind to the active site of the substrate. However, since it is not the actual substrate, there is no reaction and the substance remains in the active site, disabling the enzyme. This is a non-reversible process and the enzyme is no longer functional. The “mimic” molecule competes with the substrate for the active site.

27. Competitive Inhibition, cont. These “mimic” molecules are commonly called poisons! The pesticide DDT works in this manner. The miracle antibiotic penicillin works in the same manner. It inhibits the enzyme certain types of pathogenic bacteria use to build their cell walls. Without the functional enzyme the bacterial cell walls are defective and weak or rupture. If the bacteria survive, this makes them weak and easy targets for antibodies and our white blood cells. Penicillin has little or no effect on human cells because we don’t have cell walls, therefore no enzyme that produces cell walls!

28. Noncompetitive Inhibition This type of inhibition is common in metabolic pathways. A metabolic pathway is a series of interconnected enzymatic steps where the products of one reaction becomes the substrates for subsequent enzymatic reactions in the pathway. This process is reversible and the enzymes are undamaged by the inhibitor molecules. This process is best observed in allosteric enzymes, where the inhibitor molecules bind to the allosteric site to deactivate the enzyme. This is called negative-feedback inhibition. This form of inhibition prevents the build up of excess products and the use of energy to produce them.

29. Applications of inhibitors  Negative feedback: end point or end product inhibition  Poisons snake bite, plant alkaloids and nerve gases.  Medicine antibiotics, sulphonamides, sedatives and stimulants

30. Cell processes (e.g. respiration or photosynthesis) consist of series of pathways controlled by enzymes ABCDEFABCDEF Enzyme pathways eFeF eDeD eCeC eAeA eBeB Each step is controlled by a different enzyme (e A, e B, e C etc) This is possible because of enzyme specificity

31. End point inhibition  The first step (controlled by e A ) is often controlled by the end product (F)  Therefore negative feedback is possible ABCDEFABCDEF  The end products are controlling their own rate of production  There is no build up of intermediates (B, C, D and E) eFeF eDeD eCeC eAeA eBeB Inhibition

32. Example: Phosphofructokinase and ATP Substrate: Fructose-6-phosphate Reaction phosphofructokinase fructose-6-phosphate + ATP  fructose-1,6-bisphosphate + ADP

33. ATP is the end point  This reaction lies near the beginning of the respiration pathway in cells  The end product of respiration is ATP  If there is a lot of ATP in the cell this enzyme is inhibited  Respiration slows down and less ATP is produced  As ATP is used up the inhibition stops and the reaction speeds up again

34. The switch: Allosteric inhibition Allosteric means “other site” E Allosteric site Active site

35. Switching off  These enzymes have two receptor sites  One site fits the substrate like other enzymes  The other site fits an inhibitor molecule Substrate cannot fit into the active site Inhibitor molecule Inhibitor fits into allosteric site

36. The allosteric site- the enzyme “on-off” switch E Active site Allosteric site empty Substrate fits into the active site The inhibitor molecule is absent Conformational change Inhibitor fits into allosteric site Substrate cannot fit into the active site Inhibitor molecule is present E

37. A change in shape  When the inhibitor is present it fits into its site and there is a conformational change in the enzyme molecule  The enzyme’s molecular shape changes  The active site of the substrate changes  The substrate cannot bind with the substrate

38. Negative feedback is achieved  The reaction slows down  This is not competitive inhibition but it is reversible  When the inhibitor concentration diminishes the enzyme’s conformation changes back to its active form

39. Phosphofructokinase  This enzyme an active site for fructose-6-phosphate molecules to bind with another phosphate group  It has an allosteric site for ATP molecules, the inhibitor  When the cell consumes a lot of ATP the level of ATP in the cell falls  No ATP binds to the allosteric site of phosphofructokinase  The enzyme’s conformation (shape) changes and the active site accepts substrate molecules

40. Phosphofructokinase  The respiration pathway accelerates and ATP (the final product) builds up in the cell  As the ATP increases, more and more ATP fits into the allosteric site of the phosphofructokinase molecules  The enzyme’s conformation changes again and stops accepting substrate molecules in the active site  Respiration slows down