Science Fair Project on Enzymes

Do you want to create an amazing science fair project for your next exhibition? You are in the right place. Read the below given article to get a complete idea on enzymes: 1. Meaning of Enzymes 2. Discovery of Enzymes 3. Nomenclature and Classification 4. Structure 5. Characteristics 6. Properties 7. Inhibition 8. Mechanism 9. Constituents.

Contents:

1. Science Fair Project on the Meaning of Enzymes
2. Science Fair Project on the Discovery of Enzymes
3. Science Fair Project on the Nomenclature and Classification of Enzymes
4. Science Fair Project on the Characteristics of Enzymes
5. Science Fair Project on the Structure of Enzymes
6. Science Fair Project on the Properties of Enzymes
7. Science Fair Project on the Inhibition of Enzymes
8. Science Fair Project on the Mechanism of Enzyme Action
9. Science Fair Project on the Constituents of Enzymes

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Science Fair Project # 1. Meaning of Enzymes:

An enzyme is a specialized protein produced within an organism or cell which is capable of catalysing a specific chemical reaction. Since the enzyme acts as a catalyst, it is sometimes referred to as biocatalyst. A catalyst influences the rate of a chemical reaction, usually without undergoing any change itself. Thus, an enzyme differs from a normal catalyst.

The enzyme may participate in a reaction by combining with the substrate. Ultimately, however, it is set free. The DNA in each cell has the necessary message (blue print) for the production of all the enzymes required by it. The cell uses this information as and when necessary to produce the enzymes required to catalyze specific reactions at any point of time.

Enzymes are synthesised by living cells. But they retain their catalytic action even when extracted from cells. For example, Rennet tablets containing the enzyme rennin from the calf’s stomach have long been used for coagulating milk protein to obtain casein (cheese from milk), for various uses, including the preparation of eatables.

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Science Fair Project # 2. Discovery of Enzymes:

The term enzyme was coined by Kuhne (1878) for catalytically active substances previously called ferments. Enzymes were discovered by accident. In 1897 a German chemist Edward Buchner was preparing yeast extract for medicinal purposes. The extracts he prepared invariably turned bad.

He added some sugar to prevent spoilage. To his surprise the sugar fermented into alcohol. Pasteur had already demonstrated that living yeast cells promoted fermentation. Buchner discovered that living cells were not necessary but that yeast extract could bring about fermentation. The word enzyme was coined. It literally means ‘in yeast’ but is now used as a general name for biocatalysts.

Definition:

Mayrback (1952) defined ‘enzymes are simple or compound proteins acting a specific catalysts’. They may also be defined as ‘organic substances capable of catalysing chemical reactions in the living systems.’

However, enzymes speed up the rate of reaction without affecting the equilibrium. They exhibit a striking specificity to manifest themselves in their catalytic reactions, enhancing and controlling the course of these processes.

In 1878, Freidrich W. Kuhne coined the word enzyme (Gk. en + zyme = in yeast), and replaced the older word ‘ferment’ by it.

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Science Fair Project # 3. Nomenclature and Classification of Enzymes:

Enzymes were variously named in the past. Enzymes, such as ptyalin (salivary amylase), pepsin and trypsin give no indication of their action. While other enzymes such as amylase, sucrase, protease and lipase were named after the substrates on which they acted — amylose (starch), sucrose, protein and lipids respectively.

Still others were named according to the source from which they were obtained — papain from papaya, bromelain from pineapple (of family Bromeliaceae).

Some like DNA polymerase indicate its specific action, polymerisation. To facilitate scientific communication and to maintain uniformity, enzymes are now categorised and named by an international code of enzyme nomenclature.

The name of each enzyme ends with an ase and consists of two parts. The first part indicates its substrates and the second the reaction catalysed. For example, glutamate pyruvate transaminase transfers an amino group from the substrate glutamate to another substrate pyruvate.

Enzymes are grouped into six major classes:

Class 1. Oxireductases:

These catalyse oxidation or reduction of their substrates and act by removing or adding electrons from or to substrates, e.g., cytochrome oxidase oxidises cytochrome.

Class 2. Transferases:

These transfer Specific groups from one substrate to another. The chemical group transferred in the process is not in a free state, e.g., glutamate pyruvate transaminase.

Class 3. Hydrolases:

These break down large molecules into smaller ones by the introduction of water (hydrolysis) and breaking of specific covalent bonds. Most digestive enzymes belong to this category, e.g., amylase which hydrolyses starch.

Class 4. Lyases:

These catalyse the cleavage of specific covalent bonds and removal of groups without hydrolysis, e.g., histidine decarboxylase cleaves C─C bond in histidine to form carbon dioxide and histamine.

Class 5. Isomerases:

These catalyse the rearrangement of molecular structure to form isomeres, e.g., phosphohexose isomerase changes gulcose-6-phosphate to fructose-6-phosphate (both are hexose phosphates).

Class 6. Ligases:

The catalyse covalent bonding of two substrates to form a large molecule. The energy for reaction is derived from the hydrolysis of ATP. Pyruvate carboxylase combines pyruvate and carbon dioxide to form oxaloacetate at the expense of ATP.

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Science Fair Project # 4. Structure of Enzymes:

All enzymes are proteins but all proteins are not enzymes. Many enzymes contain a non­-protein part called the prosthetic group. These are tightly bound to the enzyme.

Some prosthetic groups are metal compounds. For example, iron-porphyrin complexes form the prosthetic groups of cytochromes. In addition, certain organic compounds and inorganic ions are required for enzyme activity. They are loosely bound to the enzyme and are called co-factors.

Nucleotides of the vitamin nicotinamide (NAD) and riboflavin (FAD) are organic co-factors or coenzymes of many oxidising enzymes of the mitochondria. In addition certain metals, especially those occurring in trace amounts, facilitate enzyme reaction. Iron (Fe⁺⁺) is a co-factor responsible for the catalytic action of catalase.

All enzymes have a specific three dimensional structure, a part of which is known as the active site. An enzyme may have more than one active site.

The active site serves as a ‘lock’ into which the reactant, commonly referred to as the substrate fits in like a ‘key’ (Fig. 1.1). The points where the substrate is bound on the active site is known as the substrate-binding site.

Substrate binding causes a lowering of the activation energy and allows the reaction to proceed (Fig. 1.2). Once the reaction is completed the enzyme releases the products and is ready to catalyse again.

A single molecule of the enzyme carbonic anhydrase, the fastest enzyme known, hyrates 36 million (36×10⁶) molecules of carbon dioxide per minute.

The catalysed reaction is 10 million (10⁷) times faster than the non-catalysed reaction.

Experiment:

Take 2 ml of hydrogen peroxide (H₂O₂) solution in two test tubes. Add a pinch of manganese dioxide (MnO₂) powder to one and a small piece of fresh liver or potato to the other.

Keep the test tubes at room temperature in summer or in a beaker of warm water (at 38°C) in winter. Bubbles of oxygen evolve in the two test tubes. Thus both manganese dioxide and a tissue enzyme (peroxidase or catalase) can catalyse the evolution of oxygen from hydrogen peroxide.

Repeat the same experiment, but use boiled and cooled manganese dioxide (MnO₂) suspension and a boiled or cooled liver/potato slice. Gas evolution still occurs in the hydrogen peroxide solution treated with boiled and cooled manganese dioxide. But the tube containing the boiled tissue slices does not evolve oxygen.

This experiment demonstrates that heat inactivates the enzyme but not the inorganic catalyst.

Science Fair Project # 5. Characteristics of Enzymes:

1. Specificity:

Each enzyme can catalyse the change of either a specific substrate or a specific group of substrates only.

The specificity for a substrate may easily be demonstrated by an experiment as follows:

Experiment:

Take 3 ml of Benedict’s solution containing cupric sulphate, m two test tubes. 1 ml of starch solution (1%) is added to one and 1 ml of sucrose solution (1%) to the other. The solutions are then boiled for a couple of minutes. No change occurs in either of the test tubes. Thus, both starch and sucrose are non-reducing carbohydrates. Neither of them can reduce Cu⁺⁺ of the reagent to Cu⁺.

Now, saliva is collected in two fresh test tubes. Starch is added to one of them and sucrose to the other. Both test tubes are kept at room temperature in summer for about one hour and in warm water (about 38°C) in winter. Benedicti’s solution is then added to both test tubes and their contents are boiled for a couple of minutes. The blue solution changes to yellow or red precipitate in the test tube containing starch.

This indicates that the enzyme salivary amylase hydrolyses starch to reducing sugars. The latter reduces Cu⁺⁺ to Cu⁺ which forms the coloured precipitate of cuprous oxide (Cu₂O). But the test tube containing sucrose shows no change. Thus, salivary amylase cannot hydrolyse sucrose. No reducing sugar is produced and the colour of Benedict’s solutions remains unchanged.

2. Optimum Temperature: 

Enzymes generally work over a narrow range of temperatures. Usually it corresponds to the body temperature of the organism. For instance, human enzymes work at the normal body temperature. Each enzyme shows its highest activity at a particular temperature.

This is called the optimum temperature. Activity declines both above and below the optimum temperature. Cold preserves the enzyme in a temporarily inactive state.

Food may be preserved for long in a frozen state because neither microbial enzyme nor enzymes in the food can act at low temperatures to cause its spoilage. As demonstrated earlier (experiment 1), very high temperatures destroy enzyme activity.

This is because proteins are denatured by heat. For this reason, few cells can tolerate temperatures above 45°C. Some heat resistant species like micro-organisms living in hot springs at temperatures close to 100°C, possess heat-resistant enzymes.

3. Optimum pH:

Each enzyme shows its highest activity at a specific pH. This is called the optimum pH. Activity declines both above and below the optimum pH. Most intracellular enzymes function best around neutral pH.

Some digestive enzymes have their optimum in the acidic or alkaline range. For example, the protein digesting enzyme pepsin found in the stomach has an optimum pH of 2.0. Another protein-digesting enzyme, trypsin, found in the duodenum, functions best in an alkaline pH of 8.5.

Experiment:

Take 2 ml starch solution (1%) in two test tubes. Some saliva is collected mixed with the contents of each tube. A few drops of dilute hydrochloric acid are added to one of the tubes. Both test tubes are then kept for one hour at room temperature in summer or in beaker containing warm water (about 38°C) in winter.

Subsequently a drop of dilute iodine solution is added to each tube. In the test tube whose contents were acidified, a blue colour develops due to the reactions of unchanged starch with iodine. But the content of the other test tube is not coloured blue with iodine, indicating that starch has been hydrolysed by salivary amylase.

This shows that amylase hydrolyses starch at salivary pH, but fails to act at a more acid pH.

In the stomach, salivary amylase swallowed with food is inactivated by the hydrochloric acid present in the gastric juice.

4. Enzyme-Substrate Complex:

Each enzyme (E) possesses a substrate-binding site on the active site in its molecule. A highly reactive enzyme-substrate complex is consequently produced. The latter almost immediately dissociates into the product or products (P) and the unchanged enzyme.

Formation of the enzyme-substrate complex is essential for catalysis. The higher the affinity of the enzyme for its substrate, the greater is its catalytic activity.

5. Effect of Substrate Concentration:

With the increase in substrate concentration (S), the velocity (V) of the enzymatic reaction rises at first. But the rise in V decreases progressively with the rise in (S). The reaction ultimately reaches a maximum velocity, which is not exceeded by any further rise in substrate concentration.

This happens because enzyme molecules are fewer than the substrate molecules. Any more increase in substrate concentration will saturate all the enzyme molecules. No enzyme is left free to bind with additional molecules of the substrate.

Michaelis Constant(K_m):

Michaelis constant (K_m) of an enzyme is the substrate concentration at which the reaction attains half its maximum velocity (Fig. 1.3). It is a measure of the affinity of the enzyme for its substrate. The lower the K_m, the higher is the substrate affinity of the enzyme.

An enzyme possesses different affinities for different substrates. Thus, its K_m value differs from substrate to substrate. Proteases can act on a variety of proteins. The K_m value of the protease will vary with the type of protein.

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Science Fair Project # 6. Properties of Enzymes:

(a) Catalytic Property:

Like inorganic catalysts the enzymes are active in very small or catalytic amounts and remain unchanged after the reaction. Only a small amount of enzyme is enough to convert large quantity of the substrate into products.

Their turnover number (k cat) i.e., the number of substrate molecules converted to product by one molecule of enzyme per second per active site may range from 0.5 to 4 x 10⁷. The turnover number for catalase has been calculated as 4 x 10⁷and is among the highest known. Lysozyme on the other extreme, has lowest value of turnover number i.e., 0.5.

(b) Specificity:

Enzymes are very specific in their action. A particular enzyme usually acts on a particular substrate to catalyse a particular type of reaction. In some cases the specificity of the enzymes may not be so strong but rather general and a particular enzyme may act on a group of substrates.

(c) Reversibility:

In most of the cases the reactions catalysed by the enzymes are reversible depending upon the requirements of the cell. But, in some cases there are separate enzymes for forward and backward reactions. Or, a reaction catalysed by a particular enzyme may not be reversible at all.

(d) Sensitiveness to Heat and Inhibitors:

(i) The enzymes are very sensitive to heat, i.e., they are themolabile. They are inactivated at very low temperatures. At very high temperatures 60°-70°C usually they are destroyed (denatured). Low molecular weight enzymes are comparatively more heat stable.

(ii) Enzymes are also sensitive to inhibitors. While some inhibitors may partially inhibit their activity, other inhibitors like poisons destroy them permanently and inhibit their activity.

(e) Colloidal Nature:

The enzymes are of colloidal dimensions and present large surfaces for reactants in water to facilitate the enzyme reaction.

Science Fair Project # 7. Inhibition of Enzymes:

1. Competitive Inhibition:

The action of an enzyme may be reduced or inhibited in the presence of a substance that closely resembles the substrate in molecular structure. Such an inhibition is called a competitive inhibition of that enzyme. Due to its closed structural similarity with the substrate, the inhibitor competes with the latter for the substrate-binding site of the enzyme (Fig. 1.4).

Consequently, the enzyme cannot participate in catalysing the change of the substrate. As a result, the enzyme action declines, e.g., the inhibition of succinate dehydrogenase by malonate, which closely resembles succinate in structure.

This may be compared to a lock jammed by a key similar to the original key. Such competitive inhibitors are often used in the control of bacterial pathogens. For instance, sulpha drugs are competitive inhibitors of folic acid synthesis in bacteria as they substitute for p-amino benzoic acid, thus preventing the next step in the synthesis.

2. Non-Competitive Inhibition:

Cyanide kills an animal by inhibiting cytochrome oxidase, a mitochondrial enzyme essential for cellular respiration. This is an example of non-competitive inhibition of an enzyme.

Here the inhibitor (cyanide) has no structural similarity with the substrate (cytrochrome) and does not bind with the substrate-binding site but at some other site of the enzyme. Thus, in non-competitive inhibition substrate binding takes place but no products are formed.

3. Feedback Inhibition:

The activities of some enzymes, particularly those which form a part of a chain of reactions (metabolic pathway), are regulated internally. Some specific low molecular weight substance, such as the product/products of another enzyme further on in the chain, acts as the inhibitor.

Such a modulator substance binds with a specific site of the enzyme different from its substrate-binding site. This binding increases or decreases the enzyme action. Such enzymes are called allosteric enzymes (Fig. 1.5).

Science Fair Project # 8. Mechanism of Enzyme Action:

Swante August Arrhenius, the Nobel Prize winner of 1903 in Chemistry, first pointed out that all the molecules in a given population do not have the same kinetic energy. Some molecules due to collisions have more energy and are energy-rich molecules while the others are energy-poor molecules.

In an ordinary chemical reaction only energy rich molecules can take part at normal temperature due to an energy barrier to reaction, hence the rate of reaction is lower. The higher is the energy barrier for a molecule, the greater is its stability or inactiveness to take part in reaction. The energy required to hurdle molecules over this energy barrier is called the energy of activation.

At higher temperature the rate of chemical reaction becomes faster because increased temperature brings about an increase in the number of activated molecules by increasing their movement and number of collisions due to thermal agitation.

However, in case of enzyme catalysed reactions the rate of reaction is optimum at normal body temperatures. It is because all the molecules (energy-rich and energy-poor) can combine with the active sites of enzyme to form enzyme substrate complex which later on breaks into enzyme and the product. Thus, the enzymes act by lowering the energy of activation of the reactions (Fig. 1.6).

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Science Fair Project # 9. Constituents of Enzymes:

a. Protein Part the Enzyme (Apoenzyme):

Major part of the enzyme consists of protein whose molecular weight may vary from few thousands to over a million.

For example:

Usually the enzymic proteins consist of only one polypeptide chain, hut sometimes there may be more than one polypeptide chains.

Enzymic proteins consist of the same 20 different types of the amino acids which constitute other proteins.

Sequence of the amino acids is specific in specific enzyme proteins. Their tertiary structure is also very specific and important for their biological activity. Loss of the tertiary structure renders the enzyme inactive. For example, in enzyme ribonuclease different folds of the polypeptide chain are held together by four disulphide linkages (S-S) to give it a compact shape (Fig. 1.7 A).

If these linkages are reduced with mercaptoethanol, the disulphide groups are converted to sulphydryl groups SH) resulting in unfolding of the polypetide chain and loss of enzymic activity (Fig. 1.7 B). Reoxidation of the protein in air results in information of the disulphide bonds and return of its enzymic activity (1.7 C).

b. Active Centre or Active Site:

Active centre or the active site is that part of the enzyme which takes part in the reaction. Specific substrate combines with it and this combination brings about the biochemical reaction.

Active centre is very specific. There may be one or more active centre on a single enzyme molecule.

It is believed that the active centre consists of amino acids residues which are very close to each other in native structure of the enzyme because of the folding of the polypeptide chain, but in fact may be far apart in the primary sequence (Fig. 1.8). This also explains why unfolding of the polypeptide chain by denaturing agents results in loss of its activity.

If the active centre is present only on one fold of the polypeptide chain and does not extend over the different folds as shown in (fig. 1.9), the unfolding of the polypeptide chain may not necessarily result in loss of enzyme activity.

In enzymes consisting of more than one polypeptide chains, the active centre may extend an individual chain or over different polypeptide chains.

The neighbouring groups of amino acids although not constituting the active centre, may also have profound influence on its specificity.

c. Prosthetic Group (Non-protein Part of the Enzyme):

Sometimes a non-protein substance is required at the active centre for enzyme activity which is bound tightly to the enzyme protein by covalent linkages and is known as prosthetic group.

The prosthetic group may consist of (a) an organic compound or (b) simple metal ions, such as Cu, Zu, Mn, Mo, etc. The organic compounds acting as prosthetic groups are usually (i) a flavin compound, such as FMN (Flavin Mono-nucleotide), FAD (Flavin Adenine Dinucleotide) (ii) heme iron, i.e., iron-porphyrin or (iii) biotin, etc.