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They generally work very rapidly – The speed of action of an enzyme is expressed by its turnover number. This is the number of substrate molecules which a molecule of the enzyme turns into product per minute. The turnover numbers of enzymes vary from 100 to several million; for the majority of cases it is approximately several thousand. One of the fastest enzymes is catalase, found in tissues where it speeds up the decomposition of H2O2 into water and oxygen; catalase has a turnover number of six million. In their speed of action enzymes are much more efficient than inorganic catalysts. The reason is that the enzyme achieves a greater lowering of the activation energy that can be brought about by inorganic catalysts.
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Enzymes are not destroyed by the reactions they catalyse and so can be used again. This is not to say that a given molecule of enzyme can be used indefinitely for enzymes are unstable and are readily inactivated by heat, acids, etc. In this respect they differ from inorganic catalysts which are completely stable and can be used over and over again indefinitely.
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An enzyme can work in either direction – Metabolic reactions are reversible and the direction in which they proceed depends on the relative amounts of substrate and products present. The reaction will proceed from left to right until equilibrium between substrate and products is reached.
A + B ↔ C
If for some reason a large amount of C happens to be present the reverse reaction occurs, C being split in A and B until again equilibrium is established. The enzyme responsible for accelerating this reaction will catalyse it in either direction depending on conditions. The enzyme has no effect on the equilibrium point; it merely speeds up the reaction until that point is achieved.
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Enzymes are inactivated (denatured) by extreme heat – This property of enzymes relates to the fact they are proteins.
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The effect of temperature on the rate of reaction of an enzyme controlled reaction. The concentration of enzyme and substrate were kept constant at all temps investigated.
The diagram above shows the effect of temperature on the rate of an enzyme controlled reaction. Up to approximately 400c the rate increases smoothly. Above this temperature the rate begins to fall off and at approximately 600c the reaction ceases altogether. This is because at high temperatures enzymes have denatured. For this reason few cells can tolerate temperatures higher than 450c. Organisms living in situations where the temperature exceeds 450c either have heat resistant enzymes or are capable of regulating their body temperature. An example would be the micro organisms which are capable of living in hot springs at temperatures of 1000c and above.
If we go back to the diagram again we can see that at a low temperature such as 100c, nearly nothing is happening, this is because molecules are moving relatively slowly. Substrate molecules will often not collide with the active site, and so bonding between substrate and enzymes doesn’t often occur. As temperature rises, the enzyme and the substrate molecules move faster. Collisions happen more frequently, so the rate of reaction increases, causing more products to be made. Also when they do collide, they do so with more energy, meaning there is an increased chance of bonds being broken and positive reactions occurring. As temperature continues to increase, the speed of enzyme and substrate molecules continue to increase. However as mentioned earlier, above a certain temperature the structure of the enzyme molecule vibrates so energetically that some of the bonds holding the enzyme in its precise 3D shape begin to break down. This is the basis of denaturing, which is often irreversible once it has begun. At first the substrate molecules will fit less well into the active site of the enzyme so the rate of reaction slows down. Eventually the substrate no longer fits at all, or it can no longer be held in the right place, long enough for the reaction to occur.
The temperature at which an enzyme works best is called its OPTIMUM TEMPRETURE.
Enzymes are sensitive to pH. Every enzyme has its own range of pH at which it functions most effectively. Most intracellular enzymes function best at or around neutral. Excessive acidity or alkalinity renders them inactive. On the other hand certain digestive enzymes prefer a distinctly acidic or alkaline environment.
pH is the measure of the concentration of hydrogen ions in a solution, the lower the pH, the higher the hydrogen ion concentration. Hydrogen ions can interact with the R groups of amino acids, affecting the way in which they bond with each other and therefore affect their 3D arrangement. A pH which is very different from the optimum pH can cause the enzyme to denature.
Enzymes are specific in the reactions they catalyse, much more so than inorganic catalysts. Normally a given enzyme will catalyse only one reaction, or type of reaction. However the degree of specifity varies from one enzyme to another. Most intra cellular enzymes only work on one particular substrate, but certain digestive enzymes work on a comparatively wide range of related substrates. Examples would be catalase that only splits hydrogen peroxide and is ineffective on any natural substance, but pancreatic lipase is less specific and will digest a variety of different fats.
How enzymes work
Enzymes work by catalysing a reaction, and increasing the rate at which chemical reactions occur. In many reactions the substrate will not be converted to a product unless it is temporarily given some extra energy. This energy is called activation energy.
To change into a product the energy of the substrate must be briefly raised, by an amount known as the activation energy. This could be done by heating the substrate. When a substrate binds to the active site of an enzyme, the shape of its molecules is slightly changed. This makes it easier to change into a product: the activation energy is lower.
Enzymes decrease the activation energy of the reaction they catalyse. They do this by holding the substrate or substrates in such a way that their molecules can react more easily. Reactions catalysed by enzymes will take place rapidly at a much lower temperature than they would without them.
Lock and Key & Induced Fit Hypothesis
For an enzyme and substrate to bind they have to fit together physically. Each enzyme has a region on its surface called the active site. This is a cleft in the protein surface where the substrate binds. It has a shape that fits the substrate like a glove fits a hand or a lock fits a key. Only substrates with a particular molecular shape will have any chance to bind effectively.
Another idea arising from the lock and key hypothesis is the induced fit hypothesis that suggests that the enzyme can change shape to suit its substrate, although it cannot change completely, it means that enzyme can flex a little, when the product(s) or substrate(s) leave the active site, the active sight realigns its self to its original form.
Enzyme Immobilization
The term "immobilized" means unable to move or stationary. And that is exactly what an immobilized enzyme is: an enzyme that is physically attached to a solid support over which a substrate is passed and converted to product.
When immobilizing an enzyme to a surface, it is most important to choose a method of attachment that will prevent loss of enzyme activity by not changing the chemical nature or reactive groups in the binding site of the enzyme. In other words, attach the enzyme but do as little damage as possible. Considerable knowledge of the active site of the enzyme will prove helpful in achieving this task. It is desired to avoid reaction with the essential binding site group of the enzyme. Alternatively, an active site can be protected during attachment as long as the protective groups can be removed later on without loss of enzyme activity. In some cases, this protective function can be fulfilled by a substrate or a competitive inhibitor of the enzyme.
The surface on which the enzyme is immobilized is responsible for retaining the structure in the enzyme through hydrogen bonding or the formation of electron transition complexes. These links will prevent vibration of the enzyme and thus increase thermal stability. The micro environment of surface and enzyme has a charged nature that can cause a shift in the optimum pH of the enzyme of up to 2 pH units. This may be accompanied by a general broadening of the pH region in which the enzyme can work effectively, allowing enzymes that normally do not have similar pH regions to work together.
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Carrier-Binding : the binding of enzymes to water-insoluble carriers
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Cross-Linking: intermolecular cross-linking of enzymes by bi-functional or multi-functional reagents.
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Entrapping : incorporating enzymes into the lattices of a semi-permeable gel or enclosing the enzymes in a semi-permeable polymer membrane
Conclusion
I will use this information to try and conclude whether immobilization affects rate of reaction of enzymes at different temperatures and pH’s.
I will first carry out some preliminary experiments to determine the effect of pH and temperature on enzymes, before studying effect f immobilization.This will give me a comparative in order to determine the true meaning of my results.
