1. Enzymes
IB HL Biology 1

2. What are enzymes?
Organic catalysts that regulate chemical reactions in living organisms
Made of proteins

3. Enzyme structure Enzymes are proteins They have a globular shape
A complex 3-D structure
At right:
Human pancreatic amylase
© Dr. Anjuman Begum
© 2007 Paul Billiet ODWS

4. Globular Proteins--Proteins with mostly beta pleated sheets
Enzyme
ex. lysozyme
sucrase
Antibodies
Hormones
ex. insulin
Na+/K+ pump
Shape is very important to help the protein bind to specific molecule

5. Globular Proteins
Include most transport proteins, enzymes, and hormones
typically water-soluble, roughly spherical and tightly folded
hydrophilic nature
polar residues = on the surface
hydrophobic residues = on the interior
many diverse structures are possible

6. Globular Protein Example 1: Hemoglobin
each subunit of hemoglobin is a globular protein with an embedded heme group. In adult humans, the most common hemoglobin protein is a tetramer consisting of four polypeptide chains
The heme group consists of an iron atom held in a ring, This iron atom is the site of oxygen binding.

7. Globular Protein Example 2: Enzyme Pepsin
a protease (protein-digesting
enzyme), which is active in the
stomach
consists of 327 amino acid
residues
has a deep cleft, the bottom of
which contains a pair of
aspartate residues on either side
of the cleft which break peptide
bonds in proteins by the addition
of water: -H to one side and -OH
to the other.

8. 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
© 2007 Paul Billiet ODWS

9. Substrate Specificity
Reactant an enzyme acts on is referred to as enzyme’s substrate.
Enzyme binds to substrate (or substrates) forming enzyme-substrate complex.
While enzyme and substrate(s) are joined, catalytic action of enzyme converts substrate(s) to product(s).

10. Substrate Specificity
Enzyme + Substrate(s)
Enzyme-substrate complex
Enzyme + Product(s)

11. Enzyme and substrate(s) fit together like a lock and a key
Specificity of an enzyme results from its shape, which is a consequence of its amino acid structure.

12. Active Site The restricted part of the enzyme that binds the substrate
Usually formed by only a few of the protein’s amino acids
As substrate enters site, interactions between substrate and enzyme’s chemical groups cause enzyme to fit even more snugly around substrate (induced fit).

13. Figure 8.16 Induced fit between an enzyme and its substrate
Active site
Enzyme
(a)
(b)
Enzyme- substrate
complex

14. Induced fit between an enzyme and its substrate
The substrate enters
the active site
The enzyme changes
shape to fit more closely
Induced Fit: New Model
shape is induced in enzyme molecule as substrate fits into cleft of active site
positions substrate to transitional state in order to react
OLD-- Lock and Key Model
The enzyme is shaped to fit the substrate.

15. 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
The lock is analogous to the enzyme and the substrate analogous to the key (Emil Fischer 1890)
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
© 2007 Paul Billiet ODWS

16. The Lock and Key Hypothesis
Enzyme may be used again
Enzyme-substrate complex E S P
Reaction coordinate
© 2007 Paul Billiet ODWS

17. The Induced Fit Hypothesis
Some proteins can change their shape (conformation)
Daniel Koshland 1958
When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation
The active site is then molded into a precise conformation--sort of like a hand in a glove
The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)
© 2007 Paul Billiet ODWS

18. The Induced Fit Hypothesis
Hexokinase (a) without (b) with glucose substrate
This explains how some enzymes can react with a range of substrates of similar types
© 2007 Paul Billiet ODWS

19. Figure 8.17 The active site and catalytic cycle of an enzyme
1 Substrates enter active site; enzyme
changes shape so its active site
embraces the substrates (induced fit).
Substrates
Products
Enzyme
Enzyme-substrate
complex
5 Products are
Released.
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.
4 Substrates are
Converted into
Products.
6 Active site
is available for
two new substrate
molecules.

20. 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
Examples of coenzymes and cofactors
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.REES
STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)
© 2007 Paul Billiet ODWS

21. Types of Chemical Reactions
Exergonic – energy released
Endergonic – energy required

22. Free energy changes (G) in exergonic and endergonic reactions
(a) Exergonic reaction: energy released
Reactants
Products
Energy
Progress of the reaction
Amount of
energy
released (∆G<0)
Free energy
required (∆G>0)
(b) Endergonic reaction: energy required
Occur spontaneously
Do NOT occur spontaneously

23. Activation Energy (EA)
Often supplied in form of heat absorbed from the surroundings
Increases speed of reactant molecules, and therefore increases number of effective collisions
Thermal agitation makes bonds of reactants more likely to break

24. Activation Energy (EA) in Biological Systems
Application of heat is inappropriate
High temperatures denature proteins and destroy cells
Heat would speed up ALL reactions, not just the necessary ones
Organisms therefore use enzyme catalysts, which lower the EA barrier

25. Denaturation: a structural change in a protein that usually results in the loss of its biological properties

26. How do enzymes function?
Enzymes lower the activation energy needed for a reaction to proceed.

27. How do enzymes function?
Reactants must absorb energy to get over EA
At transition state, bonds can be formed and broken

28. Ways Enzymes Lower EA Activation Energy Animation
Enzyme serves as template for substrates to come together in proper orientation
Enzyme stretches substrate(s) toward transition state conformation
Enzyme provides microenvironment conducive to reaction
Ex: acidic side chains on active site’s amino acids provides a low pH pocket
Direct participation of active site in reaction via brief covalent bonding with substrate.

29. Making reactions go faster
Increasing the temperature make molecules move faster
Biological systems are very sensitive to temperature changes.
Enzymes can increase the rate of reactions without increasing the temperature.
They do this by lowering the activation energy.
They create a new reaction pathway: “a short cut”
© 2007 Paul Billiet ODWS

30. Catalytic cycle
1. Substrate binds to enzyme with weak bonds. Enzyme changes shape to fit substrate.
4. Active site is ready for another substrate molecule
2. Substrate is converted to products.
3. Products are released.

31. Factors Influencing Enzyme Activity
Temperature pH
Concentration of substrate
Concentration of enzyme
Inhibitors

32. 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.
© 2007 Paul Billiet ODWS

33. Enzyme working conditions: temperature
If temperature is too high, enzyme will denature: hydrogen, ionic bonds disrupted
Activity increases to a point then slows down

34. 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
© 2007 Paul Billiet ODWS

35. Effect of temperature on enzyme activity
Low temp. – molecules move slowly
As temperature increases to optimum temperature, molecules move faster and reaction rate increases
At very high temp. – enzyme structure is denatured, and rate falls rapidly

36. Enzyme working conditions: pH
pH optima vary-some enzymes require high pH, some low
Shifts in pH can interfere with ionic bonding, hydrophobic interactions

37. 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 its substrate molecules will occur
This change in ionization will affect the binding of the substrate with the active site.
© 2007 Paul Billiet ODWS

38. Substrate concentration: Non-enzymatic reactions
Reaction velocity
Substrate concentration
The increase in velocity is proportional to the substrate concentration
© 2007 Paul Billiet ODWS

39. Substrate concentration: Enzymatic reactions
Reaction velocity
Substrate concentration
Vmax
Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied.
If you alter the concentration of the enzyme then Vmax will change too.
© 2007 Paul Billiet ODWS

40. Inhibitors (Clegg 264)
Inhibitors are chemicals or substances that reduce the rate of enzymatic 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.
© 2007 Paul Billiet ODWS

41. Types of Enzyme Inhibitors
Nonspecific inhibitors
Affect all enzymes in same manner
Produce physical and chemical changes within protein portion of enzymes
Changes in protein structure of enzymes leads to irreversible conditions, including denaturing
Temperature and pH are examples of nonspecific inhibitors

42. Types of Enzyme Inhibitors
Competitive inhibitors affect enzymes by competing with the substrate to occupy the active site
Chemical structure and molecular geometry closely resembles substrate
They interact with active site, but are not turned into products; therefore, they become “stuck” in position, preventing substrates from attaching
High substrate levels can displace these inhibitors from the active site, reversing the action of the inhibitors; this is referred to as reversible-competitive inhibition

43. Effect of competitive inhibitors on enzyme activity
Normal activity
Increasing substrate concentration →

44. Examples of Competitive Inhibitors
O2 competing with CO2 active site of Rubisco during photosynthesis
Use of ethanol in methanol poisoning
Methanol poisoning occurs because methanol is oxidized to formaldehyde and formic acid which attack the optic nerve causing blindness. Ethanol is given as an antidote for methanol poisoning because ethanol competitively inhibits the oxidation of methanol. Ethanol is oxidized in preference to methanol and consequently, the oxidation of methanol is slowed down so that the toxic by-products do not have a chance to accumulate. Source:

45. Types of Enzyme Inhibitors
Non-competitive inhibitors
Substances that attach to the enzyme other than at the active site
Net effect changes the shape of enzyme, and thus changes the active site
Excess substrate concentration will not influence the action of such inhibitors
Competitive vs. Non-competitive inhibition

47. Examples of Non-competitive Inhibitors
Often irreversible enzyme inhibitors.
Sarin: nerve gas released in Tokyo subway by terrorists in Binds irreversibly to R group of amino acid serine, in active site of acetylcholinesterase, a nervous system enzyme
Penicillin: blocks active site of enzyme bacteria use to make their cell walls.

48. 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
© 2008 Paul Billiet ODWS 48

49. Chemical Regulation of Enzyme Activity
Often essential to control of cellular metabolism
Certain chemicals selectively inhibit the function of specific enzymes
If inhibitor attaches to enzyme covalently, inhibition is irreversible
If inhibitor attaches to enzyme by weak bonds, inhibition is reversible

50. Figure 8.19 Inhibition of enzyme activity
Normal binding
(b) Competitive inhibition
A substrate can
bind normally to the
active site of an
enzyme.
A competitive
inhibitor mimics the
substrate, competing
for the active site.
Substrate
Active site
Enzyme
Noncompetitive inhibitor
(c) Noncompetitive inhibition
Competitive
inhibitor
A noncompetitive
inhibitor binds to the
enzyme away from
the active site, altering
the conformation of
the enzyme so that its
active site no longer
functions.

51. Regulation of Enzyme Activity: Competition
Competitive inhibitor has same shape as substrate
Prevents substrate from binding to active site by binding to active site itself
Example: curare curare, SSRI’s (antidepressants)SSRIs

52. Regulation of Enzyme Activity: Allosteric inhibition
Noncompetitive inhibitor binds to allosteric site--a site NOT the active site
Conformation of active site changes so substrate cannot bind
Cyanide is an example

53. Enzyme pathways A B C D E F
Cell processes (e.g. respiration or photosynthesis) consist of series of pathways controlled by enzymes
A B C D E F eF eD eC eA eB
Each step is controlled by a different enzyme (eA, eB, eC etc)
This is possible because of enzyme specificity
© 2008 Paul Billiet ODWS 53

54. End point inhibition
The first step (controlled by eA) is often controlled by the end product (F)
Therefore negative feedback is possible
A B C D E F eA eB eC eD eF
Inhibition
The end products are controlling their own rate of production
There is no build up of intermediates (B, C, D and E)
© 2008 Paul Billiet ODWS 54

55. Importance of Allostery Inhibition
A type of noncompetitive inhibition
Products of previous reactions act as
inhibitors of earlier enzymes
Binding of end product to an allosteric site changes shape of enzyme
Why is allostery helpful for the cell?
This feed back inhibition helps the cell by
allowing it to not overproduce certain
metabolic products

56. The switch: Allosteric inhibition
Allosteric means “other site”
Active site E
Allosteric site
© 2008 Paul Billiet ODWS 56

57. Switching off These enzymes have two receptor sites
One site fits the substrate like other enzymes
The other site fits an inhibitor molecule
Inhibitor molecule
Substrate
cannot fit into the active site
Inhibitor fits into allosteric site
© 2008 Paul Billiet ODWS 57

58. The allosteric site the enzyme “on-off” switch
Active site E
Allosteric site empty E
Conformational change
Substrate
fits into the active site
Inhibitor molecule is present
Substrate
cannot fit into the active site
The inhibitor molecule is absent
Inhibitor fits into allosteric site
© 2008 Paul Billiet ODWS 58

59. 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
© 2008 Paul Billiet ODWS 59

60. 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
© 2008 Paul Billiet ODWS 60

61. Feedback inhibition
The switching off of a biochemical pathway by the product of the pathway

62. Phosphofructokinase
This enzyme has 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
© 2008 Paul Billiet ODWS 62

63. 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
© 2008 Paul Billiet ODWS 63

64. 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
© 2008 Paul Billiet ODWS 64

65. Using Enzymes in Biotechnology
Biotechnology is the use of organisms or parts of organisms to produce things or to carry out useful processes
There are many ways in which enzymes, obtained from living organisms, can be used in biotechnology

66. The Use of Lactase in Lactose-free Milk Production
Lactase is an enzyme
Start with milk, ultra-pasteurize it and then add a natural enzyme, lactase. Lactase converts lactose (milk's primary carbohydrate) into glucose and galactose—simple sugars that are easily digested.

67. The Use of Lactase in Lactose-free Milk Production
Source of enzyme:
Lactase produced commercially can be extracted both from yeasts such as Kluyveromyces fragilis and Kluyveromyces lactis and from fungi, such as Aspergillus niger and Aspergillus oryzae
Use of lactase in biotechnology:
Producing lactose-free dairy products
Advantages:
More people can consume dairy products
It makes the milk products sweeter
Glucose and galactose taste sweeter than lactose
Good to get kids to get enough dairy!

68. bread and cheese making
detergents
Other Industrial Uses of Enzymes
medicines