Biochemistry Syllabus statements B.2 (Part 2)

This part of B.2 is mainly about enzymes

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Enzyme Basics

Most enzymes are proteins that act as catalysts by binding specifically to a substrate at the active site.

Example of an enzyme

Enzymes are biological catalysts that speed up reactions within the body. Enzymes are usually complex proteins.
Some examples of enzymes are:

• Amylase
• Protease
• Maltose

Enzymes have an active site where the substrate binds to and where the reaction occurs.

A simple diagram of the mechanism of an enzyme catalysed reaction. More on this later...

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ENzyme models

LOCK AND KEY
The lock and key model is the most basic model of enzymes, that is shown above.

• The enzyme is designed to work for only one substrate, for which the active site is specifically 'moulded'. The substrate is a perfect fit for the active site

INDUCED FIT
The induced fit model suggests that the enzyme plays a role in forming the shape of the active site. The intermolecular forces from the substrate help mould the shape of the active site so that the substrate fits.

Please praise my simply incredible drawing skills

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Enzyme Activity

​As enzyme activity depends on the conformation, it is sensitive to changes in temperature and pH and the presence of heavy metal ions.

EFFECTS OF PH AND TEMPERATURE ON ENZYMES

pH and temperature both have the effect of denaturing the enzyme. Denaturing just means that the active site has been damaged and can no longer function
Enzymes all have their own ranges of temperature and pH that they work at. For most enzymes, this is the temperature and pH of most organisms.
Each enzyme has a different optimum pH and temperature depending on where they operate in the body. Some examples are:

• Amylase (in the mouth)
  • pH 6.7
  • 32-37 C
• Catalase (in plants)
  • pH 7
  • 48C
• Protease (in the stomach)
  • pH 1.6
  • 50C

Heavy metal ions

Heavy metal ions act as non-competitive inhibitors. This is a HL concept from B.7 so I won't include the details in this post but the long and the short of it is that it indirectly destroys the active site.

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Enzyme activity

Deduction and interpretation of graphs of enzyme activity involving changes in substrate concentration, pH and temperature.

This is basically the kinetics of enzymes. The graphs you will come across here are graphs of Rate of Reaction against Substrate concentration. Because an enzyme can only work at a certain maximum rate, the graph curves off and a maximum rate of reaction is seen.

From this maximum, a 'half max' can also be calculated a bit like they do for half life in physics (and medicinal chem <3). This 'half max' is used to calculate a constant called the Michaelis constant (Km). Every enzyme has a different Michaelis constant.

A small Km value suggests that an enzyme only requires a small concentration on substrate to become saturated, and a large Km value suggests that an enzyme requires a large concentration of substrate to become saturated.

Long story short: The smaller the Michaelis constant, the 'worse' an enzyme is at catalysing reactions in that you need a lot more of it to reach the same rate of reaction compared to an enzyme with a larger Michaelis constant.

Effect of Ph and temperature (graphs)

The graph of Rate of Reaction against pH looks like a bell curve, with the largest value for the Rate of Reaction indicating the optimum pH

Enzyme activity plotted against pH for 2 different enzymes

The graph of Rate of Reaction against temperature looks different. This is because at low temperatures it does not denature unlike when the pH is low.

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Chromatography and Electrophoresis

Chromatography separation is based on different physical and chemical principles.

Explanation of the processes of paper chromatography and gel electrophoresis in amino acid and protein separation and identification.

Chromatography

Hopefully you remember this from GCSE. Chromatography is the separation of different substances based on their solubility in a solvent.

Chromatography also doesn't necessarily need to be done using paper, it can also be done using a gel :)

Enzyme Chromatography

An unknown mixture of enzymes can be analysed using chromatography and matching up the different 'spots' from that mixture with samples of known enzymes.

The enzymes will travel at different speeds with the solvent depending on how soluble their R group is in water. Sometimes however, some enzymes will travel exactly the same distance in a solvent. In this case, chromatography is done twice along the 2 planes of the paper.

1. A spot of the sample is put in the bottom left corner of the paper and is then placed in the first solvent.
2. Once the 1st chromatography is finished, the paper is turned 90 degrees clockwise, and placed in a second solvent.
3. The different spots can then be analysed based on their Rf values in both of the solvents.

Rf values

The Rf value is a ratio of the distance travelled by the solute and the distance moved by the solvent. 

Electrophoresis

Electrophoresis is another technique that can be used to separate and identify proteins and/or amino acids based on their Isoelectric point (pI). Electrophoresis uses a gel or paper soaked in a buffer solution to separate the amino acids. At the buffer pH (usually 6), some amino acids will form positive or negative ions based on their isoelectric points. These ions are then attracted to the electrodes in the electrophoresis setup.

Method

Electrophoresis can either be done using paper or a semi-firm gel. The samples to be tested are applied in the centre of a piece of paper or in a cavity in the gel. An electric field is applied over the gel or paper. Due to the electric field, the proteins and amino acids will seperate.

Factors affecting separation

The separation of the amino acids and proteins depends on how they interact with the gel or the paper and solvent. This can be affected by:

• The molecular weight (size) of the protein or amino acids
  • A a small, light amino acid like Lysine will interact less than a large protein like an enzyme
• The shape of the protein or amino acid (e.g. tertiary structure)
  • This is similar to molecular weight in that a bigger protein will interact more than a small, 'streamlined' amino acid.
• The charge/s on a protein or amino acid
  • The separation in electrophoresis is based on electric charge and the molecules attraction to the + and - of the electrophoresis setup. If the charge is bigger, the attraction will be bigger and the amino acid or protein will travel further.

Native and Denaturing electrophoresis

NATIVE ELECTROPHORESIS

• Enzymes and proteins are in their natural form
• Separation in electrophoresis is therefore based on their size AND shape

DENATURING ELECTROPHORESIS

• Sample is chemically or thermally denatured
  • This is done to break the intramolecular forces 'within' the proteins so that they have not tertiary structure. The protein chains are linearized.
• Separation in electrophoresis is therefore based on their size ONLY

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That's it for B.2!

Next on my list for biochem is B.7, the HL content for enzymes!

Comment if you have any questions or suggestions!