1. Introduction
The sum total of all the chemical reactions going on in cells is known as metabolism. Metabolism can be divided into 2 types, namely anabolism and catabolism. Catabolic reactions involve he breakdown of molecules and anabolic reactions involve the synthesis of molecules.
Chemical reactions of metabolism occur quickly, even at the relatively low temperatures in living things. This feature of metabolism is made possible by the actions of enzymes. Enzymes can be defined as biological catalysts which speed up a chemical reaction but remain unchanged at the end of the reaction.
2 Enzymes and Activation Energy
Every chemical reaction involves both bond breaking and bond making. Whenever the reaction rearranges the atoms and molecules, existing bonds in the reactants must be broken and the new bonds of the products must be formed. The reactants must absorb energy from their surroundings for their bonds to break and energy is released when the new bonds of the products are formed.
The initial investment of energy for starting the reaction i.e. the energy required to break the bonds in the reactants is known as the free energy of activation or activation energy. The free energy is the portion of a system’s energy that can perform work when temperature is uniform throughout the system.
The barrier of activation energy is essential to life. Proteins, DNA and other complex molecules of the cell have the potential to decompose spontaneously. These molecules exist only because at temperatures typical for cells, few molecules make it over the activation energy barrier. Occasionally, however, the barrier for selected reactions must be surmounted, or else the cell would be metabolically stagnant.
Bonds in the reactants only break when molecules have absorbed enough energy to become unstable. The activation energy is represented by the uphill portion of a “energy profile of a reaction” graph. The free energy of the reactants increase.
Activation energy is usually provided in the form of heat that the reactant molecules absorb from the surroundings. The absorption of thermal energy increases the speed of the reactants so they collide more frequently and more forcefully. Thermal agitation of the atoms in the molecules makes the bonds more likely to break.
At the summit of the graph, the reactants are in an unstable condition known as the transition state. The bonds can then break and the reaction occurs. As new bonds are formed, energy is released to the surroundings. In an exergonic reaction, a reaction that proceeds with the net release of free energy, the formation of new bonds releases more energy than was invested in the breaking of old bonds.
The formation of new bonds corresponds to the downhill part of the graph, which indicates a loss of free energy by the molecules. The difference in the free energy of the products and reactants is delta G for the overall reaction, which is negative for an exergonic reaction.
Even for an exergonic reaction, which is energetically downhill overall, the barrier of activation must be overcome before the reaction can occur. As heat is often a source of activation energy, increasing the temperature speeds up a reaction. However, high temperature kills cells. Organisms often use a catalyst.
Enzymes speeds up a reaction by lowering the barrier of activation energy so that the transition state is within reach even at moderate temperatures. Many reactions that would not ordinarily occur at the temperature of the organism do so readily in the presence of enzymes. An enzyme cannot change the delta G for a reaction. It can ouly quicken the reaction that occurs.
3. Properties of Enzymes
Most enzymes are globular proteins (polypeptide chains tightly folded to form spherical shapes), which have catalytic activity. Another class of biological catalysts is the ribozymes made of RNA. The properties of enzymes that are similar to those of inorganic catalysts are as follows.
Enzymes are effective in small amounts. The turnover number (number of substrate molecules which one molecule of enzyme turns into product per minute) of enzymes range from 100 to several million; for the majority, it is around several thousand.
Enzymes remain unchanged at the end of the reaction. They are not altered by the reaction they catalyse; they can be used repeatedly without undergoing permanent chemical change.
In reversible metabolic reactions, enzymes speed up the rate at which equilibrium is reached. Enzymes speed up the rate of both the forward and backward reactions and the rate at which equilibrium is attained. They do not alter the position of equilibrium.
Enzymes lower the amount of energy required to activate the reactants (the activation energy of the reaction).
Enzymes form transient complexes with substrate molecules, ordering them in a manner that facilitates their interaction.
The properties of enzymes that are not similar to those of inorganic catalysts (properties unique only to enzymes) are as follows.
Enzymes are extremely specific. Most enzymes are specific to one particular type or substrate and usually one isomer of that substrate (substrate or absolute specificity). Other enzymes only act on a group of similar substances; yet others will break one particular type of chemical linkage wherever it occurs (group or relative specificity).
Enzymes can be denatured by heat and they act most efficiently at an optimum temperature.
Enzymes are affected by pH and they act most efficiently at their optimum pH
Enzyme activity can be regulated by activators and inhibitors.
4. Site of Enzymes in Cells
Enzymes formed and retained in the cell are known as intracellular enzymes. Specific enzymes occur at specific sites in a cell. Soluble enzymes are found in the cytosol and inside membranous organelles such as mitochondria and chloroplasts. Insoluble enzymes are attached to the plasma membrane or to membranes of organelles. Some other enzymes are produced in the cell, packaged to be secreted from the cell and achieve their effects outside of the cell. These are known as extracellular enzymes.
5. Importance of Enzymes
Without enzymes, the reactions that occurs in living organisms would be too slow for the maintenance of life. The speed of reaction can be increased by raising the temperature but this would kill the organism by denaturing the proteins and disrupting the membranes. Enzymes enable metabolic reactions to proceed rapidly at low temperatures.
Enzymes control chemical reactions. Each individual reaction is catalyzed by a specific enzyme at a particular place within the cell. This ensures that metabolism proceeds by small steps in an orderly fashion.
Enzymes are classified as oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
6. Structure of Enzymes
Enzymes are complex 3D globular proteins, some of which have other non-protein associated molecules. While the enzyme molecule is normally larger than the substrate molecule it acts upon, only a small part of the enzyme actually comes into contact with the substrate. This region is called the active site and is typically a pocket or groove on the surface of the protein.
The active site is the region that binds the substrates (prosthetic groups if any) and contains catalytic groups that directly participate in the making and breaking of bonds. The active site is formed only by a few of the enzyme’s amino acids. With the rest of the protein molecule providing a framework that reinforces the configuration of the active site.
Most enzymes have one active site per molecule. A few enzymes have more than one active site, but this is uncommon. Catalase is unusual in having 4 active sites per molecule. Another region of the enzyme molecule, the allosteric site, is not at the active site. This is the site where small molecules bind and affect a change in the conformation of the enzyme, leading to a change in the active site to become more or less active.
An enzyme contains of 4 different categories of amino aicds.
Contact/binding residues form the binding site. They fit with the substrate molecule, while positioning it in the correct orientation for catalysis to occur. These residues determine the enzyme specificity.
Catalytic residues form the catalytic site. They act on the bonds in the substrate molecule and are responsible for the making and breaking of chemical bonds.
Binding and catalytic amino acids make up the active site of the enzyme. They are directly involved in enzyme action.
Structural residues interact to maintain the overall 3D conformation of the protein for proper functioning of the protein.
Non-essential residues are generally found on the surface of the protein. They have no specific functions.
7. Mechanism of Enzyme Action
In an enzyme-controlled reaction, the substrate molecules combine with the enzyme to form an enzyme-substrate complex. The enzyme then releases the product and returns to its original conformation.
7.1 Lock and Key Hypothesis
Proposed by Emil Fischer in 1890, this hypothesis states that in the same way as a key fits into a lock in a very precise way, so the substrate fits accurately into the active site of the enzyme
The active site of an enzyme has a distinctive configuration into which only certain specific substrates will fit. The shape of the active site and the positions of the various chemical groups within it, ensure that only those substrates with a complementary structure will combine with the enzyme.
An enzyme that displays substrate (absolute) specificity, catalyzing the reaction of only one substrate into a particular product, is likely to have a rigid active site that is best described by the lock and key model of substrate binding.
The 2 molecules forma temporary structure called the enzyme substrate complex. The products have a different shape from the substrate and so once formed, they are released from the active site, leaving it free to become attached to another substrate.
7.2 Induced Fit Hypothesis
In 1959, Daniel Koshland proposed that the active site could be modified as the substrate interacts with the enzyme. A the substrates enter the active site, it induces he enzyme to change its shape slightly so that the active site fits even more snugly around the substrate.
The amino acids that make up the active site are moulded into a precise conformation, bringing the chemical groups of the active site into positions that enhance their ability to catalyze the chemical reaction.
The enzymes that display group specificity, catalyzing reactions of structurally related products, apparently have more flexibility in their active sites and are better characterized by the induce fit model of enzyme-substrate binding. This hypothesis also explains the reversibility of enzymes better than the lock and key model, in which the bindng site is matched to either substrate or product and not both.
7.3 What happens at the active site?
The substrate is held in the active site by weak interactions. Side chains of a few of the amino acids that make up the active site catalyze the conversion of substrate into product and the product departs from the active site.
The active site may provide a conducive microenvironment to a particular type of reaction. If the active site has acidic amino acids, they may facilitate hydrogen ion transferto the substrate as the key step of catalysis. Hydrophobic amino acids create a water free zone in which non-polar reactants may react more easily.
Sometimes, brief covalent bonding between the substrate and a side chain of an amino acid of the enzyme forms. Subsequent steps of the reaction restore the side chains to their original states, so the active site is the same after the reaction as it was before.
Generally, enzymes lower activation energy and speed up a reaction by the following 2 ways. Firstly, they allow substrates to come together in close proximity and in the proper orientation at the active site such that bonds are exposed to attack.
Secondly, stressing the substrates, stretching and bending critical chemical bonds when the active site clutches the substrates with an induced fit. Since activation energy is proportional to the difficulty of breaking bonds, distorting substrates reduces the amount of thermal energy that must be absorbed to achieve a transition state.
8. Time-course of an Enzyme-catalyzed Reaction
Enzymes speed up reactions. The degree to which they do this is affected by factors such as temperature, pH, amount of enzyme and substrate. The rate of an enzyme catalyzed reaction can be compared under different conditions..
The rate of a reaction is the amount of substrate which is converted to product per unit time. There are 2 methods of measuring the rate of reaction. First is the measurement of the formation of the product. Second is the measurement of the disappearance of the substrate. The best way of measuring this depends on the particular reaction.
8.1 What happens over a period of time?
Enzyme is added to substrate at time 0. Both the substrate and enzyme move around freely in the solution. They collide with each other and, quite often, a substrate molecule collides with an enzyme molecule in such a way that it fits into the enzyme’s active site. The enzyme molecule converts the substrate into product, releases it and continues moving around until it collides with another substrate.
To begin with, there are many substrate molecules, so most of the enzyme will collide with a substrate as soon as they have released the product. Product will be formed very rapidly. Thus there is an initial steep rise in the amount of product or the initial steep decrease of the amount of substrate.
As time goes on, fewer and fewer substrates are lift, so there are less of them to collide with the enzymes. The rate at which product forms slows down. Eventually, all the substrates are changed to product and the graph showing the product formation becomes horizontal.
9. Factors affecting the Rate of an Enzyme-catalyzed reaction.
Any factor which influences an enzyme will alter the rate of the reaction that is being catalyzed. When investigating the effect of a given factor on he rate of an enzyme-catalyzed reaction, all other factors should be kept constant and at optimum levels wherever possible.
9.1 Temperature
At low temperatures near or below freezing point, enzymes are inactivated. With increased temperature, there is increased kinetic energy of the enzyme and substrate. Both move about more rapidly and therefore the probability of substrate colliding with active sites of enzymes is higher. Rate of reaction is increased.
Because they have more energy, when they collide, they are more likely to be able to overcome the activation energy barrier and form the product. The term temperature coefficient is used to express the effect of a 10oC rise in temperature on the rate of a chemical reaction. For most chemical reactions, the rate roughly doubles for every 10oC rise between 4oC and 40oC.
The temperature at which the rate of reaction occurs most rapidly is the optimum temperature. It allows the highest number of molecular collisions without denaturation. As the temperature further increases beyond the optimum temperature, rate of reaction drops sharply despite a high frequency of collisions.
The thermal agitation of the enzyme disrupts hydrogen bonds, ionic bonds and weak interactions that stabilize the 3D shape of the molecule. The 3D conformation of the enzyme is altered to the extent that the active site no longer fits the substrate. The enzyme is denatured and loses its catalytic ability.
An apparent optimum temperature for the action of the enzyme is due to the dual effects of heat, firstly on increasing the number of successful collisions between enzyme and substrate and secondly, on the stability of the enzyme itself. For many enzymes, the optimum temperature is around 40oC and denaturation occurs about 60oC.
9.2 pH
pH is a measure of hydrogen ion concentration in solution. Since the scale is logarithmic, a change in one pH point represents a 10 fold change in the hydrogen ion concentration. A change in pH in the environment of the protein alters the ionization of the acidic and basic groups of amino acids. As a result, ionic and hydrogen bonds that help to maintain the specific shape of the enzyme, are disrupted. A change in pH may also affect the charge of the R groups of key amino acids in the active site which forms temporary bonds with the substrate, thus prevents binding or catalysis of substrate.
The optimum pH is that at which the rate of reaction is at a maximum. The optimum pH for most enzymes fall in the range of 6 to 8. Exceptions include pepsin, a digestive enzyme in the stomach which works best at pH2. In contrast, alkaline phosphatase in the kidneys has an optimum pH of 10. At pH values only slightly above and below the optimum pH, there is a marked fall-off in enzyme efficiency.
Unlike the effects of heat on enzymes, the effects of pH are normally reversible at least within limits. Restoring the pH to optimum usually restores the rate of reaction. Ionization of R groups of a binding amino acids will affect the binding of substrate; ionization of R groups of catalytic amino acids will affect its catalytic ability.
9.3 Enzyme Concentration
The active site of enzymes may be used over and over again. Enzymes therefore work efficiently at very low concentrations. The turnover number for most enzymes is very high.
9.3.1 Low Enzyme Concentrations
Provided the temperature and pH are suitable for the reaction, and provided there are excess substrates, the rate of reaction is directly proportional to the enzyme concentration. Increase in enzyme concentration results in a proportional increase in the rate of reaction as more active sites are available to bind to the substrate. Thus. Enzyme concentration is limiting the rate of reaction.
9.3.2 High Enzyme Concentrations
The addition of enzyme increases the rate of reaction up to a point when any further addition of enzyme has no effect on the rate of reaction. This is represented as the plateau part on the graph. There are not enough substrate molecules to occupy all the active sites of the enzymes. The amount of substrates or other factors become limiting. In practice, enzyme concentrations that are much lower then substrate concentrations are used. Thus adding more enzyme noemally increases the rate of reaction i.e. enzyme is mostly limiting.
9.4 Substrate Concentration
9.4.1 Low Substrate Concentration
For a given amount of enzyme, the rate of an enzyme-controlled reaction increases proportionately with an increase in substrate concentration. The active sites of the enzyme are not all used; there is not enough substrate to occupy them all. Rate is limited by the amount of substrate.
9.4.2 High Substrate Concentration
As substrate concentration is increased, more active sites come into use. A point is reached where by all sites are being used; the active sites at any give moment are saturated with substrates. Additional substrate has to wait until the enzyme-substrate complex releases the products before it may enter the active site.
Increasing the substrate concentration cannot increase the rate of reaction, as the amount of enzyme is the limiting factor. At this point, the graph reaches a plateau. This value corresponds to the maximum rate Vmax that the reaction can proceed with a given concentration of enzyme in the presence of excess substrates. If additional enzyme is added to the reaction mixture, the reaction can further increase and a higher Vmax can be achieved.
Michaelis constant Km is the concentration of substrate required to make the reaction go at half Vmax.
9.5 Enzyme Inhibitors
A variety of molecules exists which can reduce the rate of an enzyme controlled reaction. These are called enzyme inhibitors. If the inhibitor attaches to the enzyme by covalent bonds, inhibition is usually irreversible. If the inhibitor binds to the enzyme by weak bonds, inhibition is reversible.
9.5.1 Reversible Inhibition
Reversible inhibitors form a relatively loose association with the enzyme, becoming detached when circumstances permit. Some occur naturally in the cell where they have a role in regulation of metabolism. The effect of this type of inhibitor is temporary and causes no permanent damage to the enzyme. Removal of the inhibitor restores the activity of he enzyme to normal. There are 2 types, competitive and nn-competitive inhibitors.
9.5.1.1 Competitive Inhibition
This occurs when a compound has a structure which is sufficiently to that of the normal substrate and is able to fit into the active site. Normally, it does not take part in the reaction, but while it remains there, it prevents the true substrate from entering the active site.
The substrate and inhibitor compete for a position in the active site and this form of inhibition is called competitive inhibition. The same quantity of product is formed because the substrate continues to use any enzyme molecules which are unaffected by the inhibitor. However, it takes a longer time to make the same amount of product.
This inhibition can be overcome when the substrate concentration is increased. This is because, as the substrate and inhibitor are in direct competition, the greater the proportion of substrate, the greater their chances of entering the active site, leaving fewer to be occupied by the inhibitor.
A characteristic feature of competitive inhibition is that if substrate concentration is increased, the rate of reaction increases. In the presence of a competitive inhibitor, the reaction rate eventually reaches the same maximum value as when no inhibitor is present i.e. Vmax remains unchanged. In competitive inhibition, half Vmax is reached at a higher substrate concentration compared to a non-inhibited reaction i.e. Km increases.
9.5.1.2 Non-competitive Inhibition
This type of inhibitor has no structural similarity to the substrate. It does not compete with the substrate for the active site, but combines with the enzyme at sites other than the active site. This interaction causes the enzyme to change its shape in such a way that the active site can no longer properly accommodate the substrate, thus catalysis cannot occur efficiently.
The non-competitive inhibitor puts a certain proportion of enzyme out of action, so the effective enzyme concentration is lowered. As such, the original maximum reaction rate cannot be achieved i.e. Vmax is lowered. An increase in substrate concentration will not reduce the effect of the inhibitor. It is a characteristic of this type of inhibition that an increase in substrate concentration does not affect the rate of reaction. The affinity of substrate for the non-inhibited enzyme is not affected thus Km is unchanged.
9.5.2 Irreversible Inhibition
Irreversible inhibitors bind permanently with enzyme molecules, leaving them permanently damaged and so unable to carry out their catalytic function. Heavy metal ions combine with sulphydryl (-SH) groups, forming strong covalent bonds and thus causing disulphide bonds to break.
Disulphide bonds help to maintain the shape of the enzyme molecule. Once broken, the structure of the enzyme becomes irreversibly altered with a permanent loss of its catalytic ability. Sulphydryl groups are very rarely located at the active sites of enzymes. In general, heavy metal ions do not compete with substrate molecules foe the active site.
9.6 Enzyme Cofactors
Of the protein-containing enzymes, some consist of protein only. Many other enzymes require non-protein substances called cofactors, for them to function efficiently.
A catalytically active enzyme-cofactor complex is called a holoenzyme. The enzymatically inactive protein resulting from the removal of the cofactor is referred to as an apoenzyme.
Cofactors may vary from simple inorganic ions to complex organic molecules. They are stable at relatively high temperatures. They may either remain unchanged at he end of the reaction or be regenerated by a later process. There are 3 types of cofactors as listed below.
9.6.1 Inorganic Ions
Activators are mineral ions that are necessary for the functioning of certain enzymes. They normally bind reversibly to the enzyme molecule. The metal ion may help to mould either the enzyme or substrate into a more suitable shape that allows an enzyme-substrate complex to be formed, or it may serve as the catalytic centre of the enzyme itself.
9.6.2 Prosthetic groups
Prosthetic groups are non-protein organic molecules that are tightly and permanently bound to the enzyme by covalent bonds, forming a conjugated protein. The best known prosthetic group is haem. It is an organic molecule with a porphyrin ring and iron (II) at its centre. Apart from its role as an oxygen carrier in haemoglobin, it is also the prosthetic group of the electron carrier cytochrome and of the enzyme catalase.
9.6.3 Coenzymes
These are organic molecules that are loosely and transiently bound to the enzyme. They may participate in reactions with 2 or more different enzymes. Many coenzymes are derived from vitamins. NAD is derived from nicotinic acid, a member of the vitamin B complex. Coenzyme functions as a carrier transferring chemical groups or atoms from the active site of one enzyme to the active site of another. NAD and other similar coenzymes work in conjunction with oxidoreductases inrespiration, their function being to transfer hydrogen from one enzyme to the next. They can then be regenerated.
10. Enzymes and the Control of Metabolism
With many thousands of different reactions occurring in a single cell, a very structured system of control of metabolic pathways is essential. If the cell were a soup of substrates, enzymes and products, the chances of particular reactants meeting would be small and the metabolic processes inefficient. Different enzymes need different conditions and it would be impossible to provide these in such an unstructured environment. A cell tightly regulates its metabolic pathways by controlling when and where its various enzymes are active.
10.1 Position of Enzymes in the Cell
Some enzymes are in solution within specific membrane-enclosed eukaryotic organelles, each with its own internal chemical environment. The organelles may also have varying conditions to suit the specific enzymes they contain. By controlling these conditions and the enzymes available, the cell can control the metabolic pathays within it.
In some cases, a team of enzymes for several steps of a metabolic pathway, is assembled together as a multi-enzyme complex. The arrangement controls the sequence of reactions, as the product from the first enzyme becomes the substrate for the adjacent enzyme in the complex, and so on, until the end product is released. Some enzyme complexes have fixed locations within the cell as structural components of particular membranes.
10.2 Control of Enzyme Synthesis and Breakdown
Protein synthesis is directly under the control of the mRNA in the cytoplasm and indirectly under the control of DNA in the nucleus. The nucleus controls and directs the growth and development of the cell and its metabolism; this is achieved by controlling which enzymes are present at any time, and therefore which chemical changes can occur. The half life of the enzyme is also important in the control of metabolism. Some of the enzymes in the cell are broken down to their constituent amino acids after a relatively brief life span (short half life).
10.3 Inactive forms of Enzymes
Another way of ensuring that the enzyme works only when required is to produce it in an inactive form and switch it on when needed. Proteolytic enzymes are usually biosynthesized as somewhat larger inactive precursors known as zymogens. If these enzymes were synthesized in their active forms, they would digest the tissues that synthesized them.
10.4 Allosteric Regulation
The word allosteric means different shapes and it is a characteristic of allosteric enzymes that they exist in 2 different forms, one active and the other inactive. The inactive form of the enzyme is shaped in such a way that the substrate will not fit into the active site. For the enzyme to become active, its shape must be altered so that the substrate will fit into the active site.
Regulatory molecules change the enzyme’s shape and function by attaching through weak bonds to an allosteric site, a specific receptor site on some part of the enzyme remote from the active site. The effect may be either inhibition or stimulation of the enzyme’s activity.
Most allosterically regulated enzymes are constructed form 2 or more polypeptide chains, or subunits. Each subunit has its own active site and allosteric sites are usually located where subunits are joined. The entire complex oscillates between 2 conformational states, one catalytically active, one inactive.
The binding of an allosteric activator to an allosteric site stabilizes the conformation that has a functional active site, thus speeding up catalysis. The binding of an allosteric inhibitor stabilizes the inactive form of the enzyme.
10.4.1 End product/feedback Inhibition
Feedback inhibition is the switching off of a metabolic pathway by its end product, which acts as an inhibitor of an enzyme within the pathway. When the end product of a metabolic pathway begins to accumulate, it may act as an allosteric inhibitor of the enzyme controlling a step in the pathway. As the end product is used up, its production is switched back on again. Thus the process is self-regulatory. The mechanism is termed as negative feedback because the information from the end of the pathway which has fed back to the start has a negative effect. Feed back inhibition thereby prevents the cell from wasting chemical resources to synthesize more product than is necessary.
10.4.2 Cooperativity
The binding of a substrate molecule to an enzyme induces a favourable change in the shape of the active site. If the enzyme has 2 or more subunits, the interaction with 1 substrate triggers the same favourable conformational change in the active sites of all the other subunits of the enzyme. This mechanism resembles allosteric activation. Cooperativity amplifies the response of enzymes to substrates. One substrate molecule primes an enzyme to accept additional substrate molecules.
