Enzymes
Topics:
1. Enzymes: Structure and Composition
2. Enzymes Classification and Nomenclature 3. Enzymatic Action and Higher Rate Energy Conversions 4. Factors Affecting Enzyme Activity 5. Co factors 6. Industrial Applications 1. Enzymes: Structure and Composition
Enzymes are the organic compounds that speed up the rate of reaction between substances. The peculiarity of the enzymes is they themselves will not be consumed in the reaction. In living cells, almost all enzymes are proteins. These specific enzymes have the ability to participate in the metabolic transformations taking place in the cells. The rate of reaction between biomolecules is increased much due to the ability of such enzymatic proteins. Some enzymes are capable of even reversing a reaction from the direction it would normally precede. There are some catalytic reactions in which there would not be any transformation of biomolecules taking place without the participation of enzymes. As such, enzymes are vital to our bodily functions as digestion and absorption which serves the necessary nutrients to the body.
Structure:
An enzyme can be easily represented by a linear diagram. Like the proteins, enzymes also possess a primary structure which depicts the amino acid sequence of a protein. Similarly it possesses the secondary and tertiary structure. In the tertiary structure of proteins, the back bone of the protein chain fold upon itself, leading to the criss crossing of the chain by itself. This folds results in the formation of many crevices or pockets. One of such pockets formed is called as the ‘active site’. The
presence of this active site makes the enzymes to catalyze the chemical reactions effectively.
An active site of an enzyme is the significant location into which the substrate fits. Thus enzymes, through their active site, catalyze reactions at a high rate. Enzymes are popularly known as ‘bio catalysts’. The catalysis process of these bio catalysts differs from that of the inorganic catalysts in a number of ways. The prime most difference is that inorganic catalysts work efficiently even at unfavorable conditions like higher temperatures and pressures, but enzymes get damaged at high temperatures even above 40 degree Celsius. However enzymes isolated from organisms like thermophilic bacteria that normally thrive under extremely high temperatures like hot vents and sulphur springs are stable. They even retain their catalytic activity at higher temperatures of 80 to 90 degree Celsius. Thus ‘thermal stability’ is an important quality of such enzymes isolated from thermophilic organisms.
Composition:
Enzymes are the biomacromolecules having molecular weights ranging from about 10,000 to over 1 million. There are some enzymes which are not or proteinaceous nature and consists of small catalytic RNA molecules. There are some nucleic acids which behave like enzymes and are termed as ‘ribozymes’. Commonly, enzymes are multiprotein complexes which are made up of a number of individual protein subunits. Many enzymes catalyze reactions individually by themselves without any assistance. There are some exceptions of enzymes which require an additional non-protein component which is termed as a coenzyme or prosthetic group. This may be an organic molecule, often a vitamin derivative, or inorganic metal ions such as Fe2+, Mg2+, Mn2+, or Zn2+ .
In most of the cases, the coenzyme participates directly in the catalytic reaction. For example, it may serve as an intermediate carrier of a group being transferred from one substrate to another.
Some enzymes have coenzymes that are tightly bound to the protein and difficult to remove, while others have coenzymes that dissociate readily. When the apoenzyme (protein moiety) and the coenzyme are separated from each other, neither of them will possess the catalytic properties of the original conjugated protein or the holoenzyme). At the same time, the fully active holoenzyme can often be reconstituted by simply mixing the apoenzyme and the coenzyme together. The same coenzyme may be associated with many enzymes which catalyze different reactions. Hence the specificity of the reaction is thus determined by the nature of the apoenzyme rather than that of the coenzyme. The complete amino acid sequence of several enzymes has been determined by chemical methods. Even the exact three-dimensional molecular structure of a few enzymes has been deduced through x-ray crystallographic methods.
2.
Enzymes Classification and Nomenclature:
There have been thousands of enzymes have been discovered, isolated and studied so far. Most of these enzymes have been classified into different groups based on the types of reaction of the catalyses.
Nomenclature:
Enzymes are often are named after the substrate involved, simply by adding ase to the name of the substrate to which it binds. For example, lactase is the enzyme that catalyzes the digestion of lactose, or milk sugar, while urease catalyzes the chemical breakdown of urea, a substance in urine. This naming strategy may result in different enzymes, called isoenzymes, with the same function having the same basic name. Isoenzymes have a different amino acid sequence and might be distinguished by their optimal pH, immunological or kinetic properties. Moreover, the normal physiological reaction of enzyme catalysis need not be the same as under artificial conditions. This can result in the same enzyme being identified with two different names. For example Glucose isomerase, is used industrially to convert glucose into the sweetener fructose, is termed as a xylose isomerase in vivo.
Classification:
Enzymes are divided into 6 classes each with 4 to 13 subclasses and named accordingly by a four digit number. The International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes, called as the EC numbers. Each enzyme is described by a sequence of four numbers preceded by "EC". The first number broadly classifies the enzyme based on its mechanism. The top-level organization of enzymes is
EC 1 Oxidoreductases: catalyze oxidation/reduction reactions EC 2 Transferases: transfer a functional group EC 3 Hydrolases: catalyze the hydrolysis of various bonds EC 4 Lyases: cleave different bonds by means other than hydrolysis and oxidation EC 5 Isomerases: catalyze isomerization changes within a single molecule EC 6 Ligases: join two molecules with covalent bonds
Enzymes are classified according to the reactions they catalyze.
The six classes are:
Oxidoreducatses / dehydrogenases: Enzymes which catalyze oxidoreduction between two substrates S and S' are known as oxidoreducatases. For example, Alcohol dehydrogenase is an oxidoreductase which converts alcohols into aldehydes or ketones. The generalized reaction is A reduced S + An oxidized S’ → A reduced S’ + An oxidized S Transferases: Enzymes catalyzing a transfer of a group B (other than hydrogen)
between a pair of substrate S and S’ are termed as Transferases. For example, Aminotransferases catalyzes the amino acid degradation by removing amino groups. The generalized reaction is S – B + S’ → S + S’ – B
Hydrolases: Enzymes catalyzing the hydrolysis of ester, ether, peptide, glycoside, C-C are called as hydrolases. C-halide and P-N bonds are hydrolases. Glucose-6phosphatase a hydrolase that removes the phosphate group from glucose-6-phosphate, leaving glucose and H3PO4. Cholinesterase, which catalyzes the hydrolysis of acetylcholine, plays an important role in the transmission of nervous impulses. Lyases: Enzymes that catalyze the removal of groups from substrates by mechanisms other than hydrolysis leaving double bonds are called as lyases. Pyruvate decarboxylase is an example of a lyase that removes CO2 from pyruvate. Isomerases: These are the enzymes which includes all the enzymes catalyzing the inter conversion of optical, geometrical or positional isomers. Ribulose phosphate epimerase is a perfect example for such an isomerase that catalyzes the interconversion of ribulose-5-phosphate and xylulose-5-phosphate.The structure if glucose isomerase is as follows:
Ligases: Ligases are the enzymes which catalyze the linking together of two compounds. For example, enzymes which catalyze joining of C- O, C- S, C- N, P – O etc bonds. Hexokinase is a ligase which catalyzes the interconversion of glucose and ATP with glucose-6-phosphate and ADP.
3.
Enzymatic Action and Higher Rate Energy Conversions:
Physical and Chemical changes:
Chemical compounds can undergo two types of changes. A physical change simply refers to the change in the shape, where no any breaking of bonds is involved. This is a typical physical process. Another type of physical process is a change in the state of matter. When ice melts into water, or when camphor sublimes into vapour, these are all considered physical processes.
Unlike this, when bonds are broken and new bonds are formed during a transformation, then such type of change is called as a chemical reaction. Mostly chemical reactions are irreversible.
Zn H 2 SO4 ZnSO4 H 2 O
This is an inorganic chemical reaction.
Reaction Rate:
Rate of a physical or chemical process is defined by the amount of product formed per unit time. Rate can also be called as velocity if the direction is specified. Rates of physical and chemical processes are influenced by among their factors. The nominal rule is that rate doubles or decreases by half for very 10 degree Celsius change in either direction. Catalytic reactions proceed at rate very faster than uncatalysed ones. When enzyme catalyst reactions are observed, the rate would be far higher than the same but uncatalysed reaction. For example,
CO 2 H 2 O Carbonic H 2 CO3 anhydrase
If any enzyme is absent, this reaction proceeds very slow about 200 molecules of H2CO3 being formed in an hour. Instead, if it involves an enzyme present within the cytoplasm called carbonic anhydrase, the reaction speed about considerably with about 600,000 molecules formed every second. The enzyme has accelerated the reaction rate about 1000 million times. It is crystal clear that the power of the enzymes is incredible. Inside living cells, there are thousands of types of enzymes each catalyzing a unique chemical or metabolic reaction.
Role of enzymes in metabolic pathways: Metabolic pathway:
A multistep chemical reaction, when each of the steps is catalyzed by the same enzyme complex or different enzymes, is called as metabolic pathway. For example, in Glycolytic pathway, glucose gives two molecules of pyruvic acid where every step is catalyzed by specific enzymes.
This is actually a metabolic pathway in which glucose is converted into pyruvic acid through the different enzyme catalyzed metabolic reactions. This metabolic pathway gives rise to a variety of end metabolic products.
Lactic acid is formed in our skeletal muscles in the absence of oxygen (i.e.) under anaerobic conditions. Under normal aerobic conditions, pyruvic acid is formed. In yeast, during the fermentation process, the same pathway leads to the production of ethyl alcohol. Thus it is understood that in different conditions, there is a possibility of different kinds of products.
Higher rate energy conversions:
In a chemical reaction, substances known as reactants interact with one another to create new substances, called products. Energy is an important component in the chemical reaction, because a certain threshold, termed the activation energy, must be crossed before a reaction can occur. There are three possible ways to increase the rate at which a reaction takes place and to hasten the crossing of the activation energy threshold. To increase the concentration of reactants To increase the temperature of the reaction To introduce a catalyst to speed up the reaction
Enzymes catalyze the chemical reactions without actually taking part in the reactions.
“Lock and Key” model:
Every specific enzyme will interact chemically with only one particular substance termed a substrate. The two parts of the enzyme and the substrate fit together, according to a widely accepted theory introduced by the German chemist Emil Fischer in the 1894, as a key fits into its lock. Each type of enzyme possesses an explicit three-dimensional shape that enables it to fit with the substrate, which has a complementary shape that of the enzyme. This concept is often referred to as "the lock and key" model. Even though this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve during their action. Enzymes bind their reactants or substrates at special folds and clefts which are named as active sites, in the structure of the substrate. An enzyme might have many active sites, since they need to catalyze a variety of reactions in the metabolic pathways. This creates an effect on their size also, so that they form very large organic compounds having atomic mass figures as high as one million amu. One atomic mass unit (amu) is approximately equal to the mass of a proton, a positively charged particle in the nucleus of an atom. Starch is the normal polysaccharide we consume in our food. This needs to be broken down into its simple monomers of glucose so that the living cells will get energy. The energy which is required to break apart the substrate is quite large.
An enzyme with the correct complementary molecular shape to the substrate molecule of the starch attaches itself to the substrate molecule, forming a chemical bond within it. The formation of these bonds causes the breaking apart of other bonds within the substrate molecule. After the completion of its work, the enzyme will move on to another uncatalyzed substrate molecule. Thus chemical reactions always involve a change in the bonds between molecules. After the expected bond breaking, making process is completed and the product is released from the active site.
Transition state:
In further simple terms, the structure of the substrate has transformed into the structure of the product. The pathways of this transformation must go through the so called ‘transition state structure’. The chemical or metabolic conversion refers to a reaction. The chemical which is converted into a product is called as a ‘substrate’. For this reason, the enzymes that are basically proteins with three dimensional structures include an active site which converts a substrate(S) into a product (P) [S → P] The substrate S has to bind the enzyme at the active site within a cleft or a pocket. In order to accomplish this, the substrate has to diffuse towards the active site. Thus this is a mandatory formation of an ES complex stands from enzyme. The complex formation is a transient phenomenon. During the state, when substrate is bound to the enzyme active site, a new structure of the substrate called as the ‘transition structure’ is formed. Each enzyme has a substrate biding site in its molecule so that a highly reactive substrate – enzyme complex is produced. This complex is short – lived and dissociates into it S products P and the unchanged enzyme with an intermediate formation of the enzyme product complex EP. The formation of the ES complex is essential for catalysis.
E S ES EP E P
The catalytic cycle of an enzyme action can be described in the following steps: 1. First and foremost, the substrate binds to the active site of the enzyme, fitting into the active site. 2. The binding site of the enzyme induces the enzyme to alter its shape, fitting more tightly around the substrate. 3. The active site of the enzyme, now in close proximity of the substrate breaks the chemical bonds and the substrate and the new enzyme – product complex is formed. 4. The enzyme will then release the products formed by the reaction. 5. The free enzyme now again binds with another uncatalyzed substrate molecule and runs through the catalytic cycle once again.
There could be many more altered structural states between the stable substrate and the end product. This fact reveals that all the other intermediate structures are unstable.
Stability of enzyme:
Stability is a phenomenon related to the energy status of the molecule or the structure. This can be clearly understood by looking at this pictorial representation through a graph which is depicted as follows:
In this graph, the y axis represents the potential energy content. The x axis represents the progression of the structural transformation or states through the transition states. Two different things can be noted: the energy level difference between S and P. If P is at lower level than S, this reaction is an exothermic reaction.
Activation energy:
Energy needs not to be supplied in the form of heating for the formation of products. However whether it is an endothermic or energy requiring process, the S has to go through such higher energy state or transition state. The difference in average energy content of S from that of their transition state is called as activation energy. Enzymes eventually bring down this energy barrier making the transition of S to P so easy.
4.
Factors Affecting Enzyme Activity:
The activity of an enzyme is affected by a change in the conditions which can alter the tertiary structure of the protein. These include the temperature, pH, and change in substrate concentration or binding of specific chemicals that regulate its activity.
Temperature:
Proteins change shape as there is a change in the temperatures. The major portion of an enzyme's activity is based on its shape. The temperature changes can mess up the process and the enzyme won't work. Each enzyme shows its highest activity at a particular temperature and pH known as the optimum temperature. Activity declines both below and above the optimum value. Low temperature preserves the enzyme in a temporarily inactive state whereas high temperature destroys enzymatic activity because heat denatures the proteins.
pH Levels:
pH is a measure of acidity. Enzymes generally function in a narrow range of pH values. Like the changes in temperature affects the enzyme, the acidity of its surrounding environment also play an important role. Every enzyme shows its highest activity at a particular pH known as the optimum pH. Activity of an enzyme turns down significantly below and above the optimum value. An increased acidity near an enzyme can cause a change in its shape. The enzyme could untangle its folds and pockets and hence become totally ineffective. The activity of an enzyme depends upon the nature of its shape or in particularly the active site of it.
Concentration of the substrate:
With the increase in substrate concentration, the velocity of the enzymatic reaction increases at first. The reaction ultimately reaches a maximum velocity of Vmax which is not exceeded by any further rise in the concentration of the substrate. This is because the enzyme molecules are fewer than the substrate molecules and after saturation of these molecules; there are no free enzyme molecules to bind with the additional substrate molecules.
Activators:
Sometimes in order to make an enzyme work faster, our body creates an activator. Activators make enzymes work harder and faster.
Inhibitors:
Inhibitors behave in an opposite manner to the activators. Inhibitors either slow down or stop the activity of an enzyme. They often bond to the protein, changing the overall shape of the enzyme. When there is a shape change, the enzyme will not work definitely in the same way. The malevolent example of an inhibitor is the snake venom.
The activity of an enzyme is also sensitive to the presence of specific chemicals that bind to the enzyme. When the binding of the chemical shuts off the enzyme activity, the process is called as ‘inhibition’ and the chemical is called as an ‘inhibitor’.
Competitive inhibition:
When the inhibitor closely resembles the substrate in its molecular structure and inhibits the activity of an enzyme, it is known as ‘competitive inhibitor’. Due to its close structural similarity with the substrate, the inhibitor competes with the substrate for the substrate binding site of the enzyme. Consequently, the substrate cannot bind and as a result, the enzyme action declines. The inhibition of the enzyme succinic dehydrogenase by malonate which closely resembles the substrate succinate in structure is a best example of this type. Such competitive inhibitors are often used in the control of bacterial pathogens.
5. Co factors:
Enzymes, in general are composed of one or more polypeptide chains. However there are a number of cases, in order to make the enzyme catalytically active, some non – protein constituents called co- factors are bound to the enzyme. Cofactors can either be inorganic like metal ions and iron-sulfur clusters or organic compounds like flavin and heme). Flavin and heme cofactors are often involved in redox reactions.
Apoenzymes:
Enzymes that require a cofactor but do not have one bound are called apoenzymes. An apoenzyme together with its cofactor(s) is called a holoenzyme which is the active form of the enzyme. Most cofactors are not covalently attached to an enzyme, but are very tightly bound.
Prosthetic Groups:
There are some exclusive organic compounds that are tightly bound to the apoenzyme and are can be easily differentiated from the other cofactors. Such organic compounds are known as ‘prosthetic groups’. These organic prosthetic groups can be covalently bound. One best example is the prosthetic group thiamine pyrophosphate which is covalently attached in the enzyme pyruvate dehydrogenase. These tightly-bound cofactors are distinguished from other coenzymes such as NADH since they are not released from the active site during the reaction. For example, in peroxidase and catalase, which catalyze the break down of hydrogen peroxide to water and oxygen, haem is the prosthetic group and it serves as a part of the active site of the enzyme.
Co –enzymes:
Co –enzymes are also organic compounds but their association with the apoenzyme is only transient, usually occurring during the course of catalysts. Also, co – enzymes serve as co – factors in a number of different enzyme catalyzed reactions. The essential chemical components of many co enzymes are vitamins. For example, coenzyme nicotinamide adenine dinucleotide (NAD) and NADP contained the vitamin Niacin or Nicotonic acid. Zinc, a popular metal ion is a co factor for the proteolytic enzyme carboxypeptidase.
Another example of an enzyme that contains Zinc ion as a cofactor is carbonic anhydrase, with a zinc cofactor bound in its active site. These tightly-bound molecules are normally found in the active site and are involved in catalysis. Carbonic anhydrase is an exclusive enzyme which catalyzes its reaction in either direction depending on the concentration of its reactants. For example, when the concentration of CO2 is higher, like inside the tissues, it catalyses the formation of H2CO3 from CO2 and H2O.
CO2 H 2 O CArbonic H 2 CO3 anhydrase
When the concentration of CO2 is lower, like inside the lungs, it catalyses the break down of H2CO3 into CO2 and H2O.
H 2 CO3 CArbonic CO2 H 2O anhydrase
When the co-factor is removed from the enzyme, the catalytic activity is lost. This proves the fact that they play a decisive role in the catalytic activity of the enzymes.
6. Industrial Application of Enzymes:
i) Brewing industry:
Barley is used for the production of malt. Enzymes from barley degrade starch and proteins to produce simple sugar, amino acids and peptides. These organic substances are further utilized by yeast for fermentation process.
ii) Production of fruit juices:
Cellulases and pectinases are the enzymes used for clarifying fruit juices.
iii) Baking industry:
Enzyme alpha-amylase catalyzes the release of simple sugars from the polymer starch. This enzyme catalyze breakdown of starch in the flour to sugar. Action of yeast on sugar produces carbon dioxide gas which is employed in the production of white bread, buns, and rolls.
iv) Starch industry:
The industrially manufactures ‘Glucose isomerase’ converts glucose into fructose in production of high fructose syrups from starchy materials. These syrups possess lower calorific values than sucrose for the same level of sweetness.
v) Baby foods:
Trypsin, the popular proteolytic enzyme is employed to predigest baby foods.
vi) Paper industry:
Enzymes like Xylanases, Cellulases and ligninases degrade starch into lower viscosity. This action helps in the sizing and coating of paper. Xylanases reduce bleach required
for decolorizing, cellulases smooth the cellulose fibre obtained from the plants, enhance water drainage, and encourages ink removal.
vii)Dairy industry:
Rennin is a specific enzyme which is derived from the stomachs of young ruminant animals like calves and lambs. This is used to hydrolyze protein and hence employed in the manufacture of cheese. Lipases are another group of enzymes implemented during the production of Roquefort cheese. Lactases are involved in the break down pf milk protein lactose into glucose and galactose.
viii) Meat industry:
Papain is the enzyme which is used to soften meat so that it will be easy for cooking.
ix) Rubber industry
Catalases are employed in rubber industry in order to generate oxygen from peroxide to convert latex into foam rubber.
x) Soap industry:
Proteases which are produced in an extracellular form from bacteria are used in the the manufacture of stain removing solutions helping with removal of protein stains from clothes. Amylases are used in the detergents for machine dish washing to remove resistant starch residues.
xi) Photographic industry:
Proteases dissolve gelatin off scrap film that allows the recovery of its silver content. xii) Molecular biology: DNA ligases, DNA polymerases and Restriction enzymes are used to manipulate DNA in genetic engineering. Also they have widespread applications in the field of forensic science.
Points to Remember:
Enzymes are the organic compounds that speed up the rate of reaction between substances. Like proteins, enzymes also possess a primary structure which depicts the amino acid sequence of a protein. An active site of an enzyme is the significant location into which the substrate fits. Enzymes with the help of their active site catalyze reactions at a high rate and are popularly known as ‘bio catalysts’.
Some nucleic acids which behave like enzymes and are termed as ‘ribozymes’. Enzymes are multiprotein complexes which are made up of a number of individual protein subunits. Enzymes are often named after the substrate involved, simply by adding ase to the name of the substrate to which it binds. For example, urease catalyzes the chemical breakdown of urea, a substance in urine.
Enzymes are divided into 6 classes each with 4 to 13 subclasses and named accordingly by a four digit number. EC 1 Oxidoreductases catalyze oxidation/reduction reactions. EC 2 Transferases transfer a functional group. EC 3 Hydrolases catalyze the hydrolysis of various bonds. EC 4 Lyases cleave different bonds by means other than hydrolysis and oxidation. EC 5 Isomerases catalyze isomerization changes within a single molecule. EC 6 Ligases join two molecules with covalent bonds. Reaction Rate of a physical or chemical process is defined by the amount of product formed per unit time. Metabolic pathway is a multistep chemical reaction, when each of the steps is catalyzed by the same enzyme complex or different enzymes. Three possible ways to increase the rate at which a reaction takes place and to hasten the crossing of the activation energy threshold are to increase the concentration of reactants, to increase the temperature of the reaction and to introduce a catalyst to speed up the reaction.
“Lock and Key” model states that every specific enzyme will interact chemically with only one particular substance termed a substrate and was proposed by German chemist Emil Fischer in the 1894, as a key fits into its lock.
The difference in average energy content of substrate from that of their transition state is called as activation energy. The activity of an enzyme is affected by a change in the conditions like the temperature, pH, and change in substrate concentration or binding of specific chemicals that can alter the tertiary structure of the protein.
When the inhibitor closely resembles the substrate in its molecular structure and inhibits the activity of an enzyme, it is known as ‘competitive inhibitor’.
In order to make the enzyme catalytically active, some non – protein constituents called co- factors are bound to the enzyme. Cofactors can either be inorganic like metal ions and iron-sulfur clusters or organic compounds like flavin and heme. Apoenzymes are enzymes that require a cofactor but do not have one bound. Prosthetic Groups are certain exclusive organic compounds that are tightly bound to the apoenzyme and are can be easily differentiated from the other cofactors.
Co –enzymes are also organic compounds but their association with the apoenzyme is only transient, usually occurring during the course of catalysts. Co – enzymes can also serve as co – factors in a number of different enzyme catalyzed reactions. Cellulases and pectinases are the enzymes used for clarifying fruit juices. Enzyme alpha-amylase catalyzes the release of simple sugars from the polymer starch. Industrially manufactured ‘Glucose isomerase’ converts glucose into fructose in production of high fructose syrups from starchy materials. Trypsin is the popular proteolytic enzyme employed to predigest baby foods. Rennin is a specific enzyme derived from the stomachs of young ruminant animals like calves and lambs and used to hydrolyze protein and hence employed in the manufacture of cheese.
Lipases are the group of enzymes implemented during the production of Roquefort cheese. Lactases are involved in the break down pf milk protein lactose into glucose and galactose. Papain is the enzyme which is used to soften meat so that it will be easy for cooking. Proteases dissolve gelatin off scrap film that allows the recovery of its silver content. DNA ligases, DNA polymerases and Restriction enzymes are used to manipulate DNA in genetic engineering and extensively used in the field of forensic science.