Enzymes Investigation

Introduction

Enzymes are proteins that speed up the rate of reaction at physiological temperatures without being used up or changed themselves. The nature of neither the end product nor the reaction equilibria is changed by the enzyme, just the rate at which it is reached. They are globular proteins, composed of polymers of amino acids.

Enzymes act by becoming part of the reaction, creating a new pathway with a lower activation energy (EA), than the than the energy needed to carry out the original reaction, as seen in the graph to the left (Campbell, 1996).

The active site of an enzyme is actually a very small proportion of the total volume. The substrate binds to the active site, forming an enzyme-substrate complex. The substrate is held in place by weak interactions such as hydrogen bonds and ionic bonds. The substrate is converted to the product and then leaves the active site, freeing it to take up another substrate molecule. As the enzyme is neither used up nor changed in the reaction it means a very small amount of enzyme can have a huge effect.

Enzymes are usually specific and catalyse on particular type of reaction or even only one specific reaction. This specificity is down to the active site, when an enzyme binds to the appropriate substrate, subtle changes in the active site occur. This alteration of the active site is known as an induced fit. Induced fit enhances catalysis, as the enzyme converts substrate to product. Release of the products restores the enzyme to its original form. The enzyme can repeat this reaction over and over, as long as substrate molecules are present.

The functioning of an enzyme can be affected by a number of factors:

Temperature

Most enzymes have a Q10 of around 2, meaning for every 10°C temperature rise the reaction rate doubles.
Most enzymes/proteins tend to be denatured or destroyed at around 40°C, but not all i.e. the enzymes of bacteria in hot springs.
The denaturation occurs due to the heat breaking bonds and causing changes in the active site so it can no longer function properly.

Enzymes usually work within a very narrow pH range.
The range is dependant on the nature of the active site; whether it accepts or donates H+ ions when binding.

Enzyme Concentration

If the substrate is always in excess then increasing the enzyme concentration will increase the rate, in a linear relationship.

Substrate Concentration

Not a linear relationship, gives a curve reaching a plateau (Vmax )
The amount of enzyme becomes the limiting factor because it has a set rate of conversion (turnover rate) that cannot be exceeded, no matter how much ...

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Enzymes usually work within a very narrow pH range.
The range is dependant on the nature of the active site; whether it accepts or donates H+ ions when binding.

Enzyme Concentration

If the substrate is always in excess then increasing the enzyme concentration will increase the rate, in a linear relationship.

Substrate Concentration

Not a linear relationship, gives a curve reaching a plateau (Vmax )
The amount of enzyme becomes the limiting factor because it has a set rate of conversion (turnover rate) that cannot be exceeded, no matter how much substrate is present
The amount of substrate needed to reach ½Vmax is called Km.
The rate can be found by using the equation:

Aims

The aim of this investigation is to study the effect of pH on the catalytic activity of the enzyme pepsin and to determine it’s optimum working pH.

We plan to do this by measuring the amount of time it takes for pepsin to break down the gelatin-binding layer on a strip of camera film in a series of buffer solutions.

Hypothesis

Pepsin will degrade the gelatin-binding layer more quickly at pH4, as this is the pH that pepsin works at in the stomach, so this must be the optimum pH.

Apparatus

6 boiling tubes

Water bath

1% Pepsin solution

pH buffers 1, 2, 3, 4, 5, 6

Stop clock

Photographic film (Ilford No4)

Scissors/craft knife and ruler for cutting film

Method

2ml of 1% pepsin solution was added to 6 boiling tubes.
To each boiling tube 2ml of a pH buffer was added. So each tube had a different buffer (1-6).
The boiling tubes were then placed into a water bath at 45oC. The tubes were then left to equilibrate for five minutes.
1 cm² of photographic film was then placed into each boiling tube and the stop clock started.
The visual appearance of the photographic film in each boiling tube was monitored and the time recorded when and if the photographic film in each boiling tube turned translucent.
To improve accuracy an average time was obtained for each pH buffer solution by the repetition of the above method.

Risk Assessment

In this investigation there are a number of risks to safety:

A first aid kit, including eyewash should be kept to hand at all times.

Results

This computer drawn graph shows that the optimum pH is just below 3, however this was not determined experimentally. From our results we can only say that the optimum pH was 3, the hand drawn graph (over) reflects this.

Discussion

The base of 35mm photographic film is celluloid and there are many layers on top of this. The most important layer is the photosensitive silver halides that react with light to produce the picture. Gelatin is a protein extracted from animal hides and used to suspend the light sensitive silver halides and bind them to the celluloid base.

Pepsin is the main protease found in the stomach of mammals and other animals. It digests proteins into smaller, peptide molecules, which can be readily absorbed by the intestinal lining. Pepsin is synthesized in an inactive form by the stomach lining; hydrochloric acid, also produced by the gastric mucosa, is necessary to convert the inactive enzyme and to maintain the optimum acidity for pepsin function.

When the photographic film is dropped into the boiling tube, the pepsin begins to break down the gelatin and the silver halides are removed from the celluloid layer making the film transparent.

The pH of a solution can have several effects of the structure and activity of enzymes.

For example, pH can have an effect of the state of ionisation of acidic or basic amino acids. Acidic amino acids have carboxyl functional groups in their side chains. Basic amino acids have amine functional groups in their side chains. If the state of ionisation of amino acids in a protein is altered then the ionic bonds that help to determine the 3-D shape of the protein can be altered. This can lead to altered protein recognition or the enzyme might become inactive. This is similar to what happens with pepsin; Pepsinogen contains an amino-terminal precursor segment of 44 residues that is proteolytically removed in the formation of Pepsin. This activation normally occurs below pH5. Cleavage of the peptide bond between leucine 16 and isoleucine 17 in the precursor segment is absolutely essential.

X-ray crystallographic analyses show that the active site is fully formed in pepsinogen but is blocked at neutral pH by residues of the precursor segment. Salt bridges are formed by six lysine and arginine side chains with carboxylate side chains of glutamate and asparate residues of the pepsin moiety when several carboxylates are protenated the pH is lowered, salt bridges between the precursor segment and the pepsin moiety are then broken. The peptide bond is now hydrolysed between the precursor and pepsin moieties (www.lancs.ac.uk, 2000). Both the structure of pepsin (top), and pepsinogen (bottom) can be seen to the left (protein data bank, -).

In general enzyme have an optimum working pH. However the optimum is not the same for each enzyme.

The pH in the stomach can vary greatly between pH1 and pH5 or more, so it would be acceptable to think that pepsin would work between these pHs. However, pepsin works in a much narrower pH range, from the table and graph we can see that the pepsin worked most effectively at pH3, and worked very slowly in only three other buffers, therefore between somewhere between ph2 and pH4 is pepsin’s optimum. This is probably closer to pH 2 as it worked faster there than at pH 4.

Further Work

To improve on this investigation we could carry out some further experiments.

Intermediate pHs should be tested to determine the exact optimum pH.
The effect of temperature should be tested so we could find the perfect conditions for pepsin.
Varying the substrate and enzyme concentrations would also give us an idea of the perfect working conditions for pepsin.
There were a number of errors in our investigation that should be thought about: