1.2.2 The Mechanism of Enzyme Action

• Within an organism there are thousands of chemical reactions happening within the cells
  • e.g. the process of photosynthesis employs a host of chemical reactions to generate a useful end-product which is glucose
• Metabolism is the sum of all the reactions happening in a cell or organism, in which molecules are synthesised (made) or broken down
• Chemical reactions need to be carefully controlled to ensure the right amount of a particular substance is made
• It is usually beneficial for the cell if chemical reactions happen quickly
  • raising the temperature can speed up chemical reactions
  • but this would speed up any unwanted reactions too
  • If the internal temperature is raised too much the cells will become damaged

Enzymes help control cell reactions

• Enzymes act as biological catalysts to speed up the rate of a chemical reaction without being changed or used up in the reaction
  • Enzymes reduce the need for high temperatures
  • They are biological as they are made in cells
• Enzymes are necessary to all living organisms as they allow all metabolic reactions to occur at a rate that can sustain life
  • For example, if we did not produce digestive enzymes, it would take around 2 - 3 weeks to digest one meal; with enzymes, it takes around 4 hours
• Different biological reactions all have a specific enzyme to help control the reaction
• Enzymes are made of protein and have a unique shape to help them function

The active site

• Chemical reactions usually involve changing a particular molecule
  • which can be split apart or joined to another molecule
  • The molecule being changed is known as the substrate
• Each enzyme has a specially shaped region known as an active site
  • The active site allows the enzyme to bind to the substrate
  • Once bound to the active site, the chemical reaction takes place
• Enzymes usually only work with one type of substrate - this is known as specificity
• The specificity of an enzyme is a result of the complementary nature between the shape of the active site on the enzyme and its substrate(s)

Enzymes are biological catalysts that work in cells, so they randomly move about wherever they are in the cell. They don’t ‘choose’ to collide with a substrate – collisions occur because all molecules are in motion in a liquid

• The ‘lock and key hypothesis’ is one simplified model that is used to explain enzyme action
• The enzyme is like a lock and the substrate is the key that fits into the active site (like a keyhole)
  • For an enzyme to work the substrate has to fit in the active site
  • If the substrate is not the correct shape it will not fit into the active site 
  • Then the reaction will not be catalysed

Diagram showing the lock and key hypothesis

Factors affecting enzyme reactions - Temperature

• Factors like temperature, pH and concentration can effect how well enzymes work
• Like any chemical reaction a higher temperature initially increases the rate of an enzyme-controlled reaction
  • The enzyme and substrate molecules have more kinetic energy, move faster and are more likely to collide
  • This leads to a faster rate of reaction
• Heating to high temperatures (beyond the optimum) will break the bonds that hold the enzyme together and the active site will lose its shape
  • the enzyme has been denatured irreversibly and will not go back into its original form
  • The substrate will not fit into the active site any more,
  • The enzyme can no longer catalyse the reaction so it stops
• Enzymes work fastest at their ‘optimum temperature’
  • In the human body, this optimum temperature is about 37⁰C which is our normal body temperature

The effect of temperature on enzyme activity

Graph showing the effect of temperature on the rate of enzyme activity

Factors affecting enzyme reactions - pH

• The pH also has an effect on enzymes, if it is too high or too low it interferes with the enzyme
• The optimum pH for most human enzymes is pH 7
  • Enzymes produced in acidic conditions, such as the stomach, have a lower optimum pH (pH 2)
  • Enzymes produced in alkaline conditions, such as the duodenum, have a higher optimum pH (pH 8 or 9)
• If the pH is too far above or too far below the optimum, the bonds that hold the amino acid chain together to make up the protein can be disrupted or broken
• This changes the shape of the active site, so the substrate can no longer fit into it, reducing the rate of activity
• Moving too far away from the optimum pH will cause the enzyme to denature and the reaction it is catalysing will stop

Effect of pH on enzyme activity

Graph showing the effect of pH on the rate of activity for an enzyme from the duodenum

Factors affecting enzyme reactions - concentration

• The greater the substrate concentration, the greater the enzyme activity and the higher the rate of reaction:
  • As the number of substrate molecules increases, the likelihood of enzyme-substrate complex formation increases
  • If the enzyme concentration remains fixed but the amount of substrate is increased past a certain point, however, all available active sites eventually become saturated and any further increase in substrate concentration will not increase the reaction rate
  • When the active sites of the enzymes are all full, any substrate molecules that are added have nowhere to bind in order to form an enzyme-substrate complex

• For this reason, in the graph below there is a linear increase in reaction rate as substrate is added, which then plateaus when all active sites become occupied
  • At this point (known as the saturation point), the substrate molecules are effectively ‘queuing up’ for an active site to become available

The effect of substrate concentration on the rate of an enzyme-catalysed reaction

Practical - The effect of pH on the rate of reaction of amylase

• Amylase is an enzyme that breaks down starch (a polysaccharide of glucose) into maltose (a disaccharide of glucose)
• The effect of different pH levels on the activity of amylase can be investigated
• The rate of reaction can be easily monitored by detecting the presence of starch
  • Starch can be detected using iodine solution 
  • If starch is present, the iodine solution will change from a brown/orange colour to blue-black

Method

• Add a drop of iodine to each of the wells of a spotting tile
• Use a syringe to place 2 cm³ of amylase into a test tube
• Add 1cm³ of buffer solution (at pH 2) to the test tube using a syringe
• Use another test tube to add 2 cm³ of starch solution to the amylase and buffer solution, start the stopwatch whilst mixing using a pipette
• Every 10 seconds, transfer a droplet of the solution to a new well of iodine solution (which should turn blue-black)
• Repeat this transfer process every 10 seconds until the iodine solution stops turning blue-black (this means the amylase has broken down all the starch)
• Record the time taken for the reaction to be completed
• Repeat the investigation with buffers at different pH values (ranging from pH 3.0 to pH 13.0) 

Investigating the effect of pH on enzyme activity

Results and Analysis

• At the optimum pH, the iodine remained orange-brown within the shortest amount of time
  • This is because the enzyme is working at its fastest rate and has digested all the starch
• At higher or lower pH's (above or below the optimum) the iodine took a longer time to stop turning blue-black or continued to turn blue-black for the entire investigation
  • This is because on either side of the optimum pH, the enzymes are starting to become denatured and as a result are unable to bind with the starch or break it down
• The time taken for the disappearance of starch is not the rate of reaction
  • It gives us an indication of the rate but it is actually the inverse of the rate
  • The shorter the time taken, the greater the rate of the reaction
• In this case, you can still calculate the rate of reaction by using the following formula:

Rate = 1 ÷ Time

• Once the rates of starch breakdown at different pH has been determined the results can be represented on a graph

A graph showing the optimum pH for an amylase in the breakdown of starch

Rate calculations for enzyme activity

• Rate calculations are important in determining how fast an enzyme is working (i.e. the rate of reaction)
• To perform a rate calculation, use the following formula:

Rate = Change ÷ Time

• 'Change' refers to the change in the substance being measured
  • This could be the amount of substrate used up in the reaction or the amount of product formed

• 'Time' refers to the time taken for that change to occur
• Another way to view the equation is as follows:

Rate = Amount of substrate used or product formed ÷ Time

Step One: Write out the equation for calculating the rate of enzyme activity

Rate = Change ÷ Time

(In this case, Rate = Amount of substrate used ÷ Time)

Step Two: Substitute in the known values and calculate the rate

Rate = 15 g ÷ 2 hours

Rate = 7.5 g / hr or 7.5 g hr⁻¹

• In the example above, the 'change' was the amount of substrate (starch) that is used up in the reaction
• In the example below, the 'change' is the amount of product that is formed in the reaction

Step One: Write out the equation for calculating the rate of enzyme activity

Rate = Change ÷ Time

(In this case, Rate = Amount of product formed ÷ Time)

Step Two: Substitute in the known values and calculate the rate

Rate = 45 cm³ ÷ 5 minutes

Rate = 9 cm³ / min or 9 cm³ min⁻¹

• Alternatively, you may not be told how much something has changed during a reaction (i.e. how much of a substrate has been used up or how much of a product has been formed)
• Instead, you may only be told the time taken for the reaction to occur
• In this case, you can still calculate the rate of reaction by using the following formula (as shown previously):

Rate = 1 ÷ Time