Biochemistry/Enzymes

Introduction

Enzymes are macromolecules that act as organic catalysts in most of the organism's biochemical reactions that have functions indispensable to maintenance and activity of life. It is crucial to note that reaction rates of certain chemical conversions occurring in living organisms are extremely low, and catalysis is necessary to maintain reasonable time of cell development and division. Majority of functional enzymes are proteins, like trypsin, fumarase or papain. A small subset of enzymes are composed of RNA and are known as ribozymes, for example hammerhead ribozyme, responsible for cleaving and joining at a specific site of RNA molecule, or 23S rRNA of prokaryotic 50S ribosome subunit activity of peptidyl transferase. The general mechanism by which enzymes work is by reducing the energy required to start the reaction, otherwise known as the activation energy. Microorganisms producing certain enzymes, or extracted and purified enzymes alone have extremely varied uses: ethanol production (Saccharomyces cerevisiae‎, Zymomonas mobilis) forensic science (restriction enzymes), and polymerase chain reaction amongst them. Uses of enzymes are not purely scientific. These diverse molecules form a huge part of industries such as brewing and pharmaceuticals; the use of enzymes alone in the brewing process of Guinness is worth over a billion euro to the Irish economy.

Structure of Enzymes

Model enzymes are monomeric, globular proteins. Majority of studies on nature of enzymatic reactions were conducted on trypsin, chymotrypsin or amylase. As every protein, to work correctly enzymes require proper folding, and, by consequence, are susceptible to deactivation by denaturation. The key to enzyme activity is a structure called active site. Interactions between residues of polypeptide chain aminoacids cause them to create a structure of defined size, shape and sequence. The difference between active site and structural domain is that the latter is able to recognize (recognition site) and process a defined molecule. Whether the interaction between the molecule and recognition site is sterical (shape-based), hydrophobic (chymotrypsin) or ionic (trypsin) it is always specific and temporal. Inhibitors often use specific covalent binding to an active site, which deactivates enzyme permanently.

Two theories regarding active sites were created. Theory of key and lock states that active site and substrate are of matching size and shape - substrate being a key and active site a lock. Binding of perfectly matching molecule would induce conformational changes of enzyme, causing catalysis. Second theory of induced fit states that active site and substrate have different, mismatching structures. When substrate meets active site it induces it to assume certain conformation, whether it is by removing structural water molecules, or straining interactions between aminoacid residues.

Mechanism of Enzymes

The mechanism how enzymes work is done by lowering the activation energy of the reaction which the specific enzyme is to catalyse. Each of the enzyme is entirely specific to the reactants; it will only work for one specific reaction, this is due to the unique active site of each enzyme. The enzyme's active site is in a unique 3D shape, with a unique pattern of electrostatic charge and hydrophobicity. Here the specific substrates -- the substances upon which the enzyme works -- are temporarily combined with the enzyme, forming an enzyme substrate complex, or ESC. The reaction then occurs, and the product of the reaction separates from the enzyme, at which point the product may be used. The enzyme is then capable of repeating the reaction many thousands of times, although they may have to be replaced every so often.

Activity of Enzymes Control of Enzyme activity in Cell The production of enzymes can be enhanced or diminished by changing the internal environment of the cell. This is called enzyme induction and inhabitation. The enzyme inhibitor or the enzyme inductor will be bound to the enzyme and either decrease or increase the activity of the enzyme. The inhibitor stops the entre of substrate to the active site. It can be reversible or irreversible. Enzyme activities are compartmentalized; this means that the reduced activity of one enzyme can lower the productivity of another, since they are linked. Some enzymes will be activated by environmental factors inside your body, for example the lowering of body pH changes the activity of hemagglutinin in influenza. Thus, the lower the potential energy barrier to reaction, the more reactants have sufficient energy and, hence, the faster the reaction will occur. All catalysts, including enzymes, function by forming a transition state, with the reactants, of lower free energy than would be found in the uncatalysed reaction.

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Enzyme Graph

Enzymes do not modify the equilibrium of a reaction. Enzymes only catalyze or speeds up the rate of reaction by stabilizing the transition state and do not participate in the chemical reaction. The transition state is the highest activation energy (Ea) on a reaction coordinate in which the molecule is only partially reacted. The activation energy is lowered when a catalyst (enzyme) is used. When an enzyme is not used, the activation energy of a reaction is much higher, which will take the reaction much longer to proceed. The enzyme is complementary to the transition state and not the substrate, in order to facilitate the reaction efficiently. A catalyst is present in the reactants and also appears in the end products.

Properties of Enzymes

1. Enzymes are sensitive to heat, and are denatured by excess heat or (inactivated by excess cold), i.e. their active site becomes permanently warped, thus the enzyme is unable to form an enzyme substrate complex. This is what happens when you fry an egg, the egg white (albumen, a type of protein, not an enzyme), is denatured. 2. Enzymes are created in cells but are capable of functioning outside of the cell. This allows the enzymes to be immobilised, without killing them. 3. Enzymes are sensitive to pH, the rate at which they can conduct reaction is dependent upon the pH of where the reaction is taking place, e.g. pepsin in the stomach has an optimum pH of about pH2. Whereas salivary amylase has an optimum pH of about 7. 4. Enzymes are reusable, and some enzymes are capable of catalysing many hundreds of thousands of reactions every second, e.g. catalase working on hydrogen peroxide. 5. Enzymes will only catalyse one reaction, e.g. invertase will only produce glucose and fructose, when a glucose solution is passed over beads of enzyme (see Immobilisation). 6. The reaction rate for an enzyme is limited by its saturation point. Saturation is essentially the point at which an enzyme can no longer speed up the reaction to compensate for the increase in substrate concentration (push the equilibrium towards the products). Graphically this is portrayed as a horizontal asymptote and is also called the maximum velocity. Each enzyme has its own maximum velocity for a given substrate. Measuring the rate of product formation with varying concentrations of substrate is one way of determining the maximum velocity of an enzyme. 7. Enzymes are capable of working in reverse, this acts as a cut off point for the amount of product being produced. If there are excess reactants the reaction will keep going and be reversed, so that there is no overload or build up of product. Enzymes are catalysts in the breaking down of substances by bonding with them briefly. Hydrogen peroxide they break it down into water and oxygen much faster than it would naturally occur. Inside the body they are used to break down food and to start off the digestion process. They are proteins they are affected by the pH level of the substance and also the temperature of the substance. The last important note is that obviously the concentration of the enzyme will affect how well it works.They are substrate specific which is illustrated by the lock and key hypothesis.

Enzymes may be immobilised in two ways, firstly by bonding them chemically to some sort of rigid structure, and secondly by physically placing the enzymes on an insoluble support structure, e.g. a gel or bead. The second method of enzyme immobilisation is much more common, is cheaper, but it does reduce the enzyme's ability to catalyse reactions.

Steps in Enzyme immobilization

1. Make up a 5% w/v solution of sodium alginate, and leave to stand overnight, to mix properly.

2. Add the enzyme, or in this case, a cell e.g. yeast, to the alginate solution, and mix in well. At this point the mixture will be very thick, and should be left for twenty minutes to eliminate the air bubbles, from the solution; such air bubbles would increase the surface area of the beads, making them less efficient.

3. Take a syringe, and fill it with the alginate/cell matrix.

4. Make a 2% w/v solution of Calcium Chloride.

5. Hold the syringe about 20 cm perpendicularly above the calcium chloride solution, and drip in the alginate solution, trying to create the smallest possible beads, approx 3-4mm in diameter.

6. Remove and discard any alginate strings or floating beads from the chloride solution.

7. Rinse the beads in deionised water.

8. Using a retort stand set up two tap funnels above each other, placing the beads in the bottom funnel, and strong glucose in the top.

9. Allow the glucose to run through the beads, and draw off the product.

The product is a type of alcohol, which should not be ingested as it is impossible to tell whether it is ethanol or methanol, the later being toxic.

Naming Enzymes

Until quite recently enzymes have been given arbitrary names, for example, ptylin (amylase), this tells us nothing of what the enzyme acts upon, instead biologists have devised a means of naming enzymes, whereby enzymes end -ase; e.g. DNA Helicase, they have also included a clue as to what the enzyme acts upon; DNA Helicase working to separate nucleotides in a chain of DNA. Other common enzymes include protease, lipases, pepsin, amylase, and lysomase.

References

Berg, Jeremy M. Biochemistry 6th Edition. New York, W. H. Freeman and Company, 2007.