Enzymes

Question 1

Where do enzymes function?

Answer 1

Enzymes function inside cells (intracellular enzymes) and outside cells (extracellular enzymes)

Question 2

What are enzymes?

Answer 2

• Enzymes are globular proteins which catalyse metabolic reactions
• They are soiled into a precise 3D shape with hydrophilic R groups on the outside of the molecule ensuring that they are soluble
• Lots of different amino acids, and active site and complex and all enzymes are globular because they can work (soluble)

Question 3

What is the lock and key hypothesis?

Answer 3

1. An active site (lock) an enzyme
2. Two which the substrate (key) of the enzyme binds to
3. The shape of the active site allows the substrate to fit perfectly
4. The idea that the enzyme has a particular shape into which the substrate fits exactly is known as the lock and key hypothesis
   - The substrate is held in place by temporary bonds which form between the substrate and some of the R groups of the enzyme’s amino acids
   - Forms the enzyme substrate complex

Question 4

What are the features of an enzyme?

Answer 4

1. The enzyme is SPECIFIC for this substrate (but some are not very specific and some can process multiple substrates
2. Not used up in reaction

Question 5

What is the induced fit hypothesis?

Answer 5

1. Enzyme molecules are more flexible that is suggested by a rigid lock and key
2. Same as lock and key but sometimes the substrate can change shape slightly as the substrate molecule enters the enzyme in order to ensure a perfect fit
3. This makes the catalysis even more efficient

Question 6

How does an enzyme catalyse a reaction?

Answer 6

1. An enzyme may catalyse a reaction in which the substrate molecule is split into two or more molecules
2. It may catalyse the joining together of two molecules, as when making a dipeptide
   - They increase the rate of reaction by lowering the activation energy of the reaction which they catalyse
   - They do this by holding the substrate or substrates in such a way that their molecules can react more easily
   - Reactions catalysed by enzymes will therefore take place rapidly at a much lower temperature than otherwise would

Question 7

What are some examples of enzymes?

Answer 7

1. Lipase (hydrolase; hydrolysis)
2. Dehydrogenase (oxidoreductase; removal or electrons or hydrogen from a substrate)
3. Kinases (transferase; transfers functional groups)

Question 8

How would you measure the rate of reaction?

Answer 8

1. Measure rate of the catalase-hydrogen peroxide reaction
   - Measure the rate at which the starch disappears from the reaction mixture
2. Take samples from mixture at known times and a dying each small to some iodine in potassium iodide solution and starch will from a blue-black colour with this solution
3. Use colorimeter measure the intensity of the blue black colour obtained and use this as a measure of the amount of starch still remaining
4. If you do this over a period of time you can plot a curve of ‘amount of starch remaining’ against ‘time’. You can then calculate the initial reaction rate (draw tangent at start?)
5. It is even easier to observe the course of this reaction if you mix starch, iodine in potassium iodide solution and amylase in a tube and take regular reading of the colour of the mixture in this one tube in a colorimeter
   - However, this is not ideal because the iodine interest with the rate of the reaction and slows it down

Question 9

How does the pH affect the rate of enzyme-catalysed reactions?

Answer 9

1. Most enzymes have an optimum pH, usually pH 7
2. If pH changes, and therefore the concentration of hydrogen ions (high in acid, low in alkali) changes
3. Hydrogen ions interact with the R groups of amino acids
4. This affects the ionic bonding between the groups which in turn affects the 3D arrangement of the enzyme molecule
5. The shape of the active site may change and therefore reduce the chances of the substrate molecule fitting into it
6. A pH which is very different from the optimum pH can cause denaturation of an enzyme

Question 10

How does a buffer solution control the pH?

Answer 10

1. Buffer solutions each have a particular pH and maintain it even if the reaction taking place would otherwise cause pH to change
2. You add a measured volume of the buffer to your reacting mixture

Question 11

How does temperature affect the rate of enzyme-catalysed reactions?

Answer 11

1. As temperature rises the the enzyme and substrate molecules move faster as they have more kinetic energy and so collisions:
   - Happen more frequently causing substrate molecules to enter the active site more often
   - They collide with more energy, and this makes it easier for bonds to be formed or broken so that the reaction can occur

Question 12

What happens to the enzyme when the temperature is too much?

Answer 12

1. Above a certain temperature, the structure of the enzyme molecule vibrate so energetically that some of the bonds holdings he enzyme molecule in its precise shape begin to break (especially true of hydrogen bonds
2. The enzyme molecule begins to lose its shape and activity and so enzyme denatured (often irreversible)
   - The temperature at which an enzyme catalyses a reaction at the maximum rate is called the optimum temperature

Question 13

What is the effect of substrate concentration on the rate of enzyme-catalysed reactions?

Answer 13

1. As substrate concentration increase, the initial rate of reaction also increases, the more substrate molecules there are, the more often an enzyme-substrate complex is made
2. However if you continue to increase the substrate concentration whilst keeping the enzyme concentration constant, there comes a point where every enzyme active site is working continuously
3. The enzyme is working at its maximum possible rate, Vmax (V for velocity)

Question 14

What is an enzyme inhibitor?

Answer 14

1. When another molecule binds to an enzyme’s active site since it is very similar shape to the enzyme’s substrate and so it inhibits the enzymes function

Question 15

What is competitive inhibition?

Answer 15

1. If an inhibitor molecule binds only briefly to the site, there is competition between it and the substrate for the site
2. If there is much more of the substrate present than the inhibitor, substrate molecules can easily bind to the active site in the usual way, and so the enzymes function is unaffected
3. However if the concentration of the inhibitor RISES, or that of the substrate FALLS, it becomes less and less likely that the substrate will collide with a empty site
4. The enzyme’s function is then inhibited

Question 16

How can you reverse competitive inhibition?

Answer 16

Competitive inhibition is said to be reversible because it can be reversed by increasing the concentration of the substrate

Question 17

What is an example of competitive inhibition?

Answer 17

• When a person has drunk ethylene glycol
  1. Ethylene glycol is rapidly converted into body to oxalic acid, which can cause irreversible kidney damage
  2. However the active site of the enzyme which converts ethylene glycol to oxalic acid will also accept ethanol
  3. Therefore if the poisoned person is given a large dose of ethanol, the ethanol acts as a COMPETITIVE INHIBITOR, slowing down the action of the enzyme on ethylene glycol for long enough to allow the ethylene glycol to be excreted

Question 18

What is non-competitive reversible inhibition?

Answer 18

1. When a molecules binds to another part of the enzyme rather than the active site
2. While the inhibitor is bound to the enzyme it can disrupt the normal arrangement of hydrogen bond and hydrophobic interactions holding the enzyme molecule in its 3D shape
3. The resulting distortion ripples across the molecule to the active site , making the enzyme unsuitable for the substrate
4. While the inhibitor is attached to the enzyme, the enzyme’s function is blocked, NO MATTER HOW MUCH SUBSTRATE IS PRESENT

Question 19

What is an example of non-competitive inhibition?

Answer 19

-In metabolic reactions must be very finely controlled and balanced so that no single enzyme can be allowed to ‘run wild’, constantly churning out more and more product

Question 20

How can you control metabolic reactions?

Answer 20

1. Use the END PRODUCT of a chain of reactions as a non-competitive reversal inhibitor
2. As the enzyme converts substrate to product, it is slowed down because the end-product binds to another part of the enzyme and prevents more substrate binding
3. However the end-product can lose its attachment to the enzyme and go on to be used elsewhere, allowing the enzyme to reform into its active state
4. As product levels fall, the enzyme is able to top them up again (this is termed END-PRODUCT INHIBITION)

Question 21

How would you investigate and explain the effect of immobilising an enzyme in alginate on its activity as compared with its activity when free in solution?

Answer 21

1. The enzyme is mixed with a solution of sodium alginate
2. Little droplets of this mixture are then added to a solution of calcium chloride
3. The sodium alginate and calcium chloride instantly react to from jelly, which turns each droplet into a little bead
4. The jelly bead contains the enzyme and the enzyme is held in the bead, or IMMOBILISED

Question 22

What is the second part to investigating the effect of immobilising an enzyme in alginate on its activity as compared with its activity when free in solution?

Answer 22

1. These beads can be packed gently into column
2. A liquid containing the enzyme’s substrate can be allowed to trickle steadily over them
3. As the substrate runs of the surface of the beads, the enzymes in the beads catalyse a reaction that converts the substrate into product
4. The product continues to trickle down the column, emerging from the bottom, where it can be collected and purified

Question 23

How can immobilising enzymes be used commercially?

Answer 23

1. Enzyme lactase can be immobilised using alginate beads
2. Milk is then allowed to run through the column containing lactase beads
3. The lactase hydrolysis the lactose in the milk to glucose and galactose
4. The milk is therefore lactose-free and can be used to make lactose-free dairy products for people who cannot digest lactose

Question 24

What are the advantages of using immobilising enzymes?

Answer 24

1. You can keep re-using the enzymes
2. The product is enzyme-free
3. Immobilised enzymes are more tolerant of temperature changes and pH changes than enzymes in solution

Question 25

What is the Vmax?

Answer 25

• The theoretical maximum rate of the reaction it catalyses

- At Vmax all the enzyme molecules are bound to the substrate molecules (the enzyme is saturated with substrate)

Question 26

How would you plot a graph to find out Vmax and Km?

Answer 26

1. Plot 1/substrate conc. on x axis and 1/velocity on y axis (double reciprocal plot)
2. 1/Vmax is the y-intercept, because this is where we know 1/[S] is zero (and therefore [S] is infinite)
3. -1/Km is the x intercept

Question 27

What is the Km?

Answer 27

• The Michaelis-Menten constant
• It is the substrate concentration at which an enzyme works at half its maximum rate (1/2 Vmax)
• At this point half the active sites of the enzyme are occupied by a substrate

Question 28

Compare different Z and E?

Answer 28

1. Z is a competitive inhibitor and E is a non-competitive inhibitor
2. Z is complementary to active site
3. E binds to allosteric site
4. E changes shape of active site
5. Effect of z is reduced by increases substract conc

Question 29

Why might something have a high turnover rate?

Answer 29

1. Molecules within cells move about eery wackily by diffusion over short distances, with tens of thousand of collision per second occurring between enzyme and substrate molecules

Question 30

What is Vmax?

Answer 30

The theoretical maximum rate of an enzyme-controlled reaction, obtained when all the active sites are occupied

Question 31

How is Vmax measured?

Answer 31

1. Reaction rate measured at different substrate concentration while keeping the enzyme concentration constant
2. As substrate concentration is increase, reaction rate rises until the reaction reaches it maximum rate Vmax

Question 32

What curve is plotted for Vmax determination?

Answer 32

1. The initial rate for each substrate concentration is plotted against substrate concentration, producig a asymptotic curve, which never fully flattens out and in theory it does at infinite substrate concentration but impossible measure and therefore to accurately read off value of Vmax
2. Therefore plot 1 /substrate concentration on x axis and 1/velocity on y axis

Question 33

What is an advantage of double reciprocal plot?

Answer 33

• 1/inifitiy is zero so Vmax can be found accurately
• Satright line
• Y axis intercept is 1/Vmax
• X axis intercept is -1/Km

Question 34

What happens the higher the affinity of the enzyme for the substrate?

Answer 34

• The lower the substrate concntration needed for happen
• So Km is a measure of affinity for its substrate
• Higher the affinity, lower Km, and quicker the reaction will reach its maximum rate, although maximum rate not affected by Km

Question 35

What are Km and Vmax?

Answer 35

1. Vmax gives information about the maximum rate of reaction that is possible (through not necessarily the rate under cell conditions)
2. Km measures the affinity of the enzyme for the substrate
   - The higher the affinity the more likely the product will be formed when a substrate molecule enter the active site rather than the substate leaving the active site again before a reaction takes place

Question 36

What does the value of Km for a particular enzyme depend on?

Answer 36

1. Identity of substrate
2. Temperature
3. pH
4. Presence of particular ions
5. Overall ions concentration
6. Presence of potions, pollutants or inhibitors

Question 37

What is the significance of Vmax and Km?

Answer 37

1. It enables scientists to make computerised models of biochemical pathways or even the behaviours of whole cells because it helps to predict how each reaction in a proposed pathway will rpcohed and therefor show the enzymes will interact. The consequences of changing conditions such as temperature, pH or the presence of inhibitors can be built into the models
2. An enzyme’s preference for different substrates can be compared quantitatively
3. By understanding what affects enzyme efficiency, scientists may in future be able to design better catalysts, linking this to genetic engineering
4. For commercially important enzyme, the performance of the same enzyme from different organisms can be compared
5. The calculation can be applied to other fields of biochemistry such as antibody-antigen binding
6. Knowing Km means the promotion of active sites occupied by substrate molecules can be calculated for any substrate concentration

Question 38

What are immobilised enzymes?

Answer 38

Enzymes that have even fixed to a surface or within ahead of agar gel