All enzymes are globular proteins, each with a specific three- dimensional tertiary structure that determines its function. Enzymes are usually specific to the substrate they work with and so the shape of the enzyme’s active site where the substrate binds has to be complementary to the shape of the substrate.
In the case of this reaction, the enzyme (amylase) is catalysing the breakdown of the substrate (starch)
Whether it is induced fit or the lock and key which occurs, the result is the same: once the enzyme has served its purpose, it is released from the product.
As previously mentioned, in order for an enzyme-substrate complex to form, the shapes of the active site and the substrate have to be complementary. The shape of the active site is determined by the tertiary structure which is the three- dimensional arrangement of polypeptides. This arrangement is stabilised by ionic, disulphide and weak hydrogen bonds.
Enzymes are affected by:
Temperature: As the temperature increases the rate of reaction will increase, because the kinetic energy of the substrate and enzyme will increase therefore resulting in more collisions, and more enzyme-substrate complexes formed. When the temperature reaches above the optimum temperature of 37oc - 40oc bonds that stabalise the enzymes tertiary structure will be broken. The enzyme will lose its shape and the active site will be altered. The substrate can no longer bind with the enzyme to form an enzyme- substrate complex
PH: Extremely low or high PH will result in a complete loss of enzyme activity as the ionic and hydrogen bonds that stabalise the enzymes tertiary structure will be broken. The enzyme will lose its shape and the active site will be altered. The substrate can no longer bind with the enzyme to form an enzyme- substrate complex. The PH will need to be around the optimum temperature of the enzyme which is usually PH7.
Concentration: If the substrate concentration is increased the rate of reaction will increase as there will be an increase in collisions which will result in an increase in enzyme- substrate complexes. As you carry on increasing the concentration of the substrate the rate of reaction reaches a maximum and no longer increases as the enzyme concentration becomes a limiting factor.
If the enzyme concentration is increased the rate of reaction will also increase, this is because substrate concentration is rarely a limiting factor.
The optimum temperature for amylase is 400c and the optimum PH is 7
Apparatus:
- Pasteur glass pipette
- Stop clock
- Stirrer
- Thermostatically controlled water bath
- Boiling tubes
- Well
Method:
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Set a thermostatically controlled water bath at 40oc
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Using a Pasteur glass pipette, pipette 4cm3 of starch of a standard concentration (0.1cm3) into a boiling tube
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Add 4cm3 of PH 6 buffer solution to the starch.
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In a separate boiling tube pipette, using a Pasteur glass pipette 2cm3 of amylase
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Pre incubate both of the boiling tubes into the 400c thermostatically controlled water bath for 5 minutes using a stop clock.
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Add the amylase to the starch, an leave in the 40oc thermostatically water bath
- Stir the solution
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Every 20 seconds using a Pasteur pipette place 0.1cm3 of the solution into a well
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Add iodine(0.01cm3) to the solution in the well
- Repeat this until the solution doesn’t turn blue/black
- About 15 results should be taken for each PH
- Then repeat all the above for PH 7, 8
Explanation:
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The temperature needs to be 370c because this is the temperature when the enzymes work the best. If the temperature is too high for the enzymes the tertiary structure and ionic bonding in the enzymes can be altered, which cause a change in the active site and the substrate will no longer fit. The rate of reaction will decrease affecting the rate at which starch is broken down into glucose.
- Leaving the substrate and enzyme to equilibrate is important because if one of the 2 is not at the right temperature the overall temperature will be affected, this will make the results inaccurate as the rate of reaction will be affected.
- The same volume of substrate and enzyme is needed for each replication, because in one of the replications if the amount of amylase was less there will be fewer enzymes to form an enzyme substrate complex and break the starch down into glucose.
- The concentration of starch and amylase would need to be the same in each replication, because if the concentration is high in one replication the rate of reaction will increase, and if it was low the rate of reaction will decrease.
- Boiled amylase will be used as a control to ensure that amylase is breaking the starch down into glucose
- The type of starch needs to be the same because the amylopectin may be easier to hydrolyse than the Amylose, as the amylopectin has a bigger surface area.
- The solution will need to be stirred to make sure that the enzyme is not at the top and the substrate at the bottom, because the enzymes will not collide with the substrate to form an enzyme-substrate complex, therefore the rate of reaction will decrease.
- Iodine will be added to the solution to show the decrease in starch, because the end point will be when the solution stays brown, this will indicate that there is no starch in the solution.
- The iodine will need to be standardised because a colour change might not be detected, therefore will not know if there is starch present, the end point will cause anomalies.