Download 2: Enzymes

enzymes
Cell structure & componentry
Cell membrane: Sit of gas transfer, contains channel pro-
Enzyme activity and structure
teins and Na and K pumps. Consists of a phospholipid bilayer.
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Nucleus: Contains an organisms DNA, and is chemically isolated from the rest of the cell.
Mitochondria: Where ATP is produced from glucose in
Enzyme activity is usually measured in mol min-1, as a measure
of how fast the substrate is metabolised into the products.
Enzymes are amino acids, with primary, secondary and tertiary
structure:
respiration.
Cytoplasm: Where the majority of the cell’s chemical reac-
Primary: The order in which the amino acids are linked, by
peptide groups.
tions take place, containing many compounds in solution.
Plant cells also have a solid cell wall, chloroplasts and a sap
vacuole.
Extracting Enzymes
Samples must be kept in a cool buffer solution to prevent disintegration of enzymes.
Firstly, the cells must be broken down in homogenization; in
tougher cells such as plant cells, a sharp blade might be used in
a food processor style contraption. Homogenization produces a
mixture of membranes, mitochondria, ribosomes, cytoplasm and
nuclei known as homogenate.
The homogenate is then separated by centrifuging it at various
speeds to separate the larger from the smaller organelles.
Finally, to obtain pure enzymes, chromatographic techniques
such as column chromatography can be used to separate
proteins based upon their size. Often multiple techniques are
combined to isolate the pure enzyme.
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Secondary: The folding into either ∝- helices or ß- pleated
sheets. Maintained by hydrogen bonding.
Tertiary: The overall 3D structure of the enzyme. Maintained
by bonds between and within side groups, such as ionic bonds
in non-neutral solutions (-CO2 and NH3 groups act as acids or
bases), covalent disulphide bonds in the amino acid cysteine,
and van der Waals and dipole-dipole interactions.
It is also important to observe the behaviour of polar and nonpolar side groups. Polar groups are hydrophilic, and so lie on
the outside of the protein, whilst non-polar, hydrophobic groups
tend to lie on the inside, insulated from water.
How enzymes work
Enzymes are able to overcome very mild conditions of temperature and pH to catalyse reactions that would normally never
take place under standard conditions. This is due to the role of
the active site.
The active site contains specific groups which form weak, transient bonds with substrates, weakening their own bonds,
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enzymes
They often act as proton acceptors or donators, and the shape
of the enzyme often changes slightly in the formation of the
enzyme-substrate complex.
Thus the specific fit of the active site is very important, and any
alteration of this can render the enzyme useless.
Factors affecting enzyme activity
• pH
»»Due to the presence of CO2H and NH3 groups, which act
as acids and bases respectively, altered pH can cause the
chemical composition of the active site to change and the
function of the enzyme to be affected.
»»Most enzymes work best at pH5-9, but some, such as
pepsin, which acts in the hydrochloric acid filled stomach,
works best at pH2.
to; they are specific to the inhibitor. This results in a change in
the shape of the active site, preventing catalysis occurring.
Allosteric inhibition is reversible, and if the inhibitor departs,
normal function is resumed. THESE CAN BE DISPLACED IF
INHIBITOR CONCENTRATION FALLS LOW.
Immobilizing enzymes
Advantages:
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To allow continuous use for long periods of time
Or repeated use in a batch process
Higher enzyme concentrations can be used
Shorter reaction times
Less need for purification of the product
You can keep the enzyme forever!
Less unwanted side products
Adsorption onto a solid
Such as porous glass. Protein is held in place by ionic bonds
between charged groups of the amino acids and charged
groups on the solid, and van der Waals. Easily produced but it’s
relatively easy for the enzyme to be washed off the support.
Trapping the enzyme
In a substance such as collagen gel. A solution containing the
enzyme and a monomer is allowed to polymerize, forming a
mesh which allows substrates to move in and out but doesnt
allow enzymes to leave. Inexpensive and hard wearing but
restricted to small substrate molecules.
Covalent binding onto a solid support
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Temperature
»»Up to a point, enzyme activity increases with increasing
temperature
»»But beyond a certain temperature, enzymes are denatured, losing 3D shape and the ability to function as
catalysts. Denaturation is irreversible.
Inhibition
Irreversible inhibitors
Bind covalently to the enzyme, disrupting the shape of the active site. They are often metal ions such as silver and mercury.
NO AMOUNT OF SUBSTRATE CAN DISPLACE THEM
Such as cellulose or nylon, which react covalently with -CO2H,
-NH2 and -OH side groups. Stabilises the enzyme with the active
site readily accessible, but rather expensive to produce suitable supports.
Encapsulation behind permeable membrane
Such as a nylon bag, which allows substrate in and out whilst
retaining enzyme. Very easy to obtain product free from enzyme, but can be quite expensive.
Producing fructose syrup
This is achieved by hydrolysing glucose into fructose, which is
far sweeter, and so less calorific. Starch from corn --> glucose,
then an enzyme from bacteria such as glucose isomerase, immobilised, is used to convert glucose into fructose in continuous reactors, relatively cheaply.
Competitive inhibitors
These have a similar shape to the substrate, and simply
outcompete the substrate. An example is malonate. They are
reversible; IF SUBSTRATE CONCENTRATION IS HIGH ENOUGH,
THEY CAN BE DISPLACED.
Allosteric inhibitors
Separating chiral forms
Only the L-amino acids are biologically useful, so the D-amino
acid aer removed using an anzyme from the Aspergillus fungi,
which is immobilized by adsorption onto a polysaccharide, and
is continuously produced, despite reduced enzyme activity, at
only 60% of the cost of an equivalent batch process.
Some enzymes have separate sites which an inhibitor can bind
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