Enzymes

Mode of action of enzymes

a) explain that enzymes are globular proteins that catalyze metabolic reactions

 An enzyme is a biological catalyst that accelerates metabolic reactions but remains unchanged at the end of the
reaction.
 Enzymes are globular proteins as they have a roughly spherical shape and are water soluble. Coiled in precise 3D
shapes with hydrophilic R groups.

b) state that enzymes function inside cells (intracellular enzymes) and outside cells (extracellular enzymes)

 Enzymes functioning inside a cell are intracellular, but those that are secreted by cells and catalyse reactions
outside cells are described as extracellular. (e.g. digestive enzymes in the gut)

c) explain the mode of action of enzymes in terms of an active site, enzyme/substrate complex, lowering of activation
energy and enzyme specificity (the lock and key hypothesis and the induced fit hypothesis should be included)

 Enzymes have specific active sites that are complementary to the shape of the substrate. The substrate is held
in place at the active site by weak hydrogen and ionic bonds. The combined structure is called the enzyme-
substrate complex.
 Activation energy is the energy required in any chemical reaction to break the bonds in reactant molecules so
that new bonds are formed to make the product
 An enzyme lowers the activation energy required for the reaction. However, overall energy released during
reaction is maintained.
 When a substrate binds to the active site of an enzyme, the shape of its molecule is slightly changed. This makes
it easier to change the substrate into a product; the activation energy is lower.
 lock and key hypothesis: The idea that the enzyme has a particular shape into which the substrate fits exactly.
 The shape of the active site is very precise and substrates that are not complementary to the shape of the active
site cannot bind. The enzyme-substrate complexes formed enable the reaction to take place more easily.
 Each type of enzyme will usually act on only one type of substrate molecule. This is because the shape of the
active site will only allow one shape of molecule to fit. The enzyme is said to be specific for this substrate.
  Induced fit hypothesis: the enzyme’s active site is not initially an exact fit to the substrate molecule.
 The enzyme molecules are more flexible and can change shape slightly as the substrate enters the enzyme.
 This means that the enzyme molecule will undergo conformational changes as the substrate combines with
enzyme’s active site, forming the enzyme-substrate complex.
 This makes the catalysis even more efficient.
 Lysozyme is a natural defence against bacteria that is found in tears, saliva and other secretions. It breaks the
polysaccharide chains that form the cell walls of bacteria.

d) investigate the progress of an enzyme-catalyzed reaction by measuring rates of formation of products (for example,
using catalase) or rates of disappearance of substrate (for example, using amylase)

 Course of a reaction: Initially, there’s a large number

of substrates and every enzyme has a substrate in its
active site.
 The rate at which the reaction occurs depends only
on how many enzymes there are and the speed at
which the enzyme can convert the substrate into
product, release it, and then bind with another
substrate.
 However, overtime, there are fewer substrates to
bind with enzymes; the reaction gets slower, until it
eventually stops.
 The rate of an enzyme-controlled reaction is always
fastest at the beginning.
 The effect that enzymes have on the rate of
reactions can be measured in two ways:
 By measuring the amount of product accumulated over a period of time. Rate of reaction = volume of product
produced/ time e.g. enzyme catalase breaking hydrogen peroxide to H2O + o2
 By measuring the rate at which the reactants disappear from the reaction mixture, the effect of the enzyme on
the rate of reaction can be determined.
 E.g.: measuring the rate at which starch disappears when the enzyme amylase is added.
 Factors that affect enzyme action

a) investigate and explain the effects of the following factors on the rate of enzyme-catalyzed reactions:

 Temperature: As the temperature increases, the kinetic energy and

the enzyme activity increases as there’s more collisions until optimal
temperature is reached (usually 40C).
 At optimal temperature, maximum rate of reaction is achieved.
 If the temperature continues to increase beyond optimal
temperature, the rate of the reaction begins to decrease as more
kinetic energy breaks the hydrogen bonds in the secondary and
tertiary structure of enzyme.
 This changes the shape of the enzyme and its active site and causes
the substrate to no longer fit. The enzyme is denatured.

 pH: Any change in the pH value of the medium around the enzyme will
cause ionic and hydrogen bonds to be damaged, this will change the 3-D
shape of the enzyme and deform the active site.

 The substrate will therefore not be able to fit into active site so the
reaction slows down or stops.

 The effects of pH are reversible within certain limits but if the pH is far
from optimal value, the enzyme gets denatured.

 Enzyme concentration: As the concentration of enzymes is increased, there are more available active sites for
substrates to fit into.
 More enzyme-substrate complexes are formed, more products are formed and the rate of reaction is increased.
 The limiting factor is the enzyme concentration. Once all substrates have formed enzyme-substrate complexes, a
further increase in concentration will have no effect on the rate of reaction.
 At this point, the limiting factor is the substrate concentration. During comparison, look at initial rate to ensure
differences in reaction rate are caused only by differences in enzyme concentration.

 Substrate concentration: As the concentration of the substrates increases, there are greater chances of collision
with enzyme.

 More enzyme-substrate complexes are formed, more products are

formed and the rate of reaction is increased.

 The limiting factor is the substrate concentration. Once all enzymes are
occupied and working at maximum rate (Vmax), a further increase in
substrate concentration will have no effect on the rate of reaction. At this
point, the limiting factor is the enzyme concentration.
 Inhibitor concentration: Inhibitors interfere with enzyme activity and reduce the rate of an enzyme catalyzed
reaction. Therefore, as the concentration of inhibitors increases, the rate of reaction decreases.

b) explain that the maximum rate of reaction (Vmax) is used to derive the Michaelis-Menten constant (Km) which is used
to compare the affinity of different enzymes for their substrates

 Turnover number: Maximum no. of molecules of substrate that an enzyme can convert to product per catalytic site
per unit time
 A typical enzyme molecule can convert around one thousand substrate molecules into product per second. This is
known as the turnover rate.
 Enzyme kinetics:
 The rate at which an enzyme catalyzes a reaction and how catalysis is affected by factors such as substrate
concentration
 The kinetics of a reaction can be measured by the appearance of product or disappearance of substrate.
 Saturation: a point beyond which increasing substrate concentration will not change the rate of reaction at all as
molecules of enzymes are engaged in catalysis
 Vmax: maximum rate of reaction when all the enzyme catalysis sites are filled with substrate
Turnover Number = Vmax / Et

 Michaelis–Menten constant (Km): The Michaelis–Menten constant is the substrate concentration at which an
enzyme works at half its maximum rate (½Vmax). At this point half the active sites of the enzyme are occupied by
substrate.
 The higher the affinity of the enzyme for the substrate, the lower the substrate concentration needed for this to
happen
 Thus, the Michaelis–Menten
constant is a measure of the
affinity of the enzyme for its
substrate.
 The higher the affinity of the
enzyme for the substrate, the
lower the substrate concentration
needed for this to happen. The
higher the affinity, the lower the
Km and the quicker the reaction
will proceed to Vmax.

 The significance of Vmax and Km values

 It enables scientists to make computerized models of biochemical pathways or even the behaviour of whole cells
because it helps to predict how each reaction in a proposed pathway will proceed and therefore how the
enzymes will interact. The consequences of changing conditions such as temperature, pH or the presence of
inhibitors can be built into the models.
 An enzyme’s preference for different substrates can be compared quantitatively.
 By understanding what affects enzyme efficiency, scientists may in future be able to design better catalysts,
linking this to genetic engineering.
 For a commercially important enzyme, the performance of the same enzyme from different organisms can be
compared.
 The calculations involved can be applied to other fields of biochemistry, such as antibody–antigen binding.
 Knowing Km means the proportion of active sites occupied by substrate molecules can be calculated for any
substrate concentration.
 Asymptotic curve
 A curve that never really flattens out in practice. In theory with infinite substrate
concentration it can.
 Makes it impossible to get a Vmax from graph.

c) explain the effects of inhibitors, both competitive and noncompetitive, on the rate of enzyme activity

 Competitive, reverse inhibitor

 Has similar shape to the substrate and fits the active site
 Reduces the number of enzyme-substrate complexes formed and
the rate of reaction decreases
 If an inhibitor molecule only briefly binds to the site there is
competition between the inhibitor and substrate.
 It is said to be reversible because it can be reversed by increasing
the concentration of the substrate.
 If the quantity of substrates increases the competition will
decrease and the rate of reaction will increase

 Non-competitive inhibitor
 Attaches themselves not to the active site of the enzyme but
elsewhere on the enzyme molecule.
 They alter the shape of the enzyme in such a way that the active site
no longer accommodates the substrate.
 They are non-competitive as they are not fighting for the active sight
 Such inhibitors can disrupt the normal arrangement of h-bonds and
hydrophobic interactions holding the enzyme molecule in shape.
 Increase in substrate concentration will not reduce the effect of the
inhibitor
 E.g.- cyanide is a non-competitive inhibitor that attaches to cytosome and stops respiration.
*irreversible inhibitor: some inhibitors can permanently bind to the active site and block the substrate. E.g.-
antibiotic penicillin works by permanently blocking the active site necessary for synthesis of bacteria cell walls.

 Feedback/end-product inhibitor
 Used to control metabolic reactions via non-competitive
reversible inhibitors.
 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.
 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.
d) investigate and explain the effect of immobilizing an enzyme in alginate on its activity as compared with its activity
when free in solution

 Enzymes are expensive. One of the best ways of keeping costs down is to use immobilized enzymes.
Process:
 Enzymes are added to solution of sodium alginate
 Droplets of this mixture are added to a CaCl solution.
 The sodium alginate and calcium chloride instantly react to form jelly, which turns each droplet into a little bead.
 (sodium ions are displaced by calcium to for calcium alginate which is insoluble)
 The jelly bead contains the enzyme. The enzyme is held in the bead, or immobilised.
 These beads can be packed gently into a column. A liquid containing the enzyme’s substrate can be allowed to
trickle steadily over them.
 As the substrate runs over the surface of the beads, the enzymes in the beads catalyse a reaction that converts
the substrate into product.
 The product continues to trickle down the column, emerging from the bottom, where it can be collected and
purified.
Advantages:
 Reusable
 Enzymes do not contain contaminated product
 Immobilised enzymes are more tolerant to changes like change in PH
 Substrate can be easily passed through many times