ENZYMES

Enzymes are proteinaceous, biocatalysts.

First enzyme discovered by Buchner.

Term enzyme was given by Kuhne.

Zymase (from yeast) was the first discovered enzyme. (Buchner)

The first purified and crystalized enzyme was urease (by J.B. Sumner) from Canavalia/Jack Bean (Lobia plant).

Proteinaceous nature of enzyme was established by Northrop and Sumner.

DEFINITION

Enzymes are biocatalysts made up of proteins (except ribozyme), which increases the rate of biochemical reactions by lowering down the activation energy.

First discovered ribozyme was L19 RNAase by T.Cech from rRNA of a protozoan Tetrahymena thermophila and RNAase P or Ribionuclease P by Altman in prokaryotic cell (Nobel prize).

GENERAL PROPERTIES OF ENZYMES

Large sized biomolecules, colloid nature with high molecular weight.

Large size (equal to colloid particles) provide, more surface area so passes large no. of active site. Large number of substrate converted into product by one molecule of enzyme at a time.

Highest molecular weight is of enzyme pyruvate dehydrogenase complex (46 lakh) participate in link reaction of respiration.

Proteinous nature

Monomer unit of a enzyme is Amino acid.

Amino acids linked togather to form polypeptide chain.

Enzymes are polypeptide chains.

Most of enzymes arrange in tertiary structure of protein or globular proteins except isoenzyme (Quaternary st.).

Tertiary structure of protein provides stability and water soluble nature to enzymes.

Synthesis of enzymes occurs on ribosomes under the control of genes.

According to one gene one polypeptide hypothesis, if a enzyme is made up of same kind of polypeptide chains then synthesize under control of same gene and if made up of different kinds of polypeptide chains then synthesized under the control of different genes. e.g., Rubisco, cytochrome, oxidase, Nitrogenase.

Specificity

Enzymes are specific for pH, temperature and substrate.

pH - The common pH range of enzymes activity is 6 - 8.

Every enzyme works on specific pH, Pepsin-2.5 pH, Hydrolase-4-5.

Rubisco, Pepcase-8.5 pH, Trypsin - 8.5 pH.

Temperature

Common range of temperature for enzyme activity is 20º – 40°C.

Enzymes works on body temperature of organism not on environmental temperature.

Enzymes of plants are affected by evironmental temperature change as plants does not show homeostasis.

At low temperature enzymes become functionally inactive, at high temperature denatured.

Substrate

Every enzyme works on specific substrate.

Substrate binds at active site of enzyme which is made of specific sequence of amino acids and recognise it’s substrate.

Ex. succinic dehydrogenase acts on succinic acid while pyruvate dehydrogenase acts on pyruvic acid.

Enzymes increase the rate of reaction by decreasing activation energy.

Activation Energy - Minimum amount of energy more than the free energy of reactents required to reach the transiation state of chemical reaction or to undergo the chemical reaction.

Turn Over Nubmer (T.O.N.) -

The number of reactent moleules converted into product by one molecule of enzyme in unit time

Highest T.O.N is of carbonic anhydrase (360 lakh / minute)

CO2 + H2O H2CO3

KM constant -

Enymes follows the Michaelis-Menten reaction kinetics.

It represents the substrate concentration at which rate of enzymetic reaction becomes the half of maximum velocity or rate.

If a enzyme passes high km constant then it’s affinity towards substrate is low and rate reaction is also low.

The energy required for a chemical reaction to proceed is called Activation energy.

The enzymes lower the activation energy. (Remember that enzymes cannot start the chemical reaction)

With the increase in concentration of substrate the enzymatic velocity also increases. At a certain value all the active site of the enzyme-molecules are saturated and the increase in substrate concentration does not increase the velocity of the enzymatic reaction.

(The concentration of substrate at which the velocity of enzymatic action reaches half of its maximum value, is called Km value or Michaelis constant).

 

Higher is the affinity of an enzyme for a substrate the lower is its Km value, i.e.

 

Ki constant (Enzyme inhibitor complex dissociation constant)

The substrate concentration at which enzyme inhibitor complex dissociate and reaction becomes normal

It is applicable only for competitive reversible inhibitions.

STRUCTURE OF ENZYME

Simple enzymes

They are made up of only protein. eg. pepsin, trypsin.

Conjugated enzymes

They are made up of protein & non protein part.

Co-enzymes - Co-enzymes are non-protein, orgainc groups, which are loosely attached to apoenzymes. They are generally made up to vitamins.

Prosthetic group - When non-protein part is tightly or firmly attached to apoenzymes.

Metal activators/co-factros/metallic factor :- Lossely attached inorganic co-factor eg. Mn, Fe, Co, Zn, Ca, Mg, Cu

Active site :

The part of polypeptide chain made up of specific sequence of amino acids at which specific substrate is to be binded and catalysed, known as active site. Very specific sequence of amino acids, at active site is determined by genetic codes.

Allosteric site :

Besides the active site's some enzymes posess additional sites, at which chemical other than substrate (allosteric modulators) are bind. These sites are known as allosteric sites and enzyme with allosteric sites are called as allosteric enzymes. e.g. hexokinase, phosphofructokinase.

 

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TERMINOLOGY

Endoenzymes - Enzymes which are functional only inside the cells. Ex. Enzymes of metabolism.

Exoenzymes - Enzymes catalysed the reactions outside the cell Eg:- enzymes of digestion, some enzymes of insectivorous plants, Zymase complex of fermentation.

Proenzyme/Zymogen - These are precursor of enzymes or inactive forms of enzymes.eg. Pepsinogen, Trypsinogen etc.

Isoenzymes - Enzymes having similar action, but little difference in their molecular configuration are called isoenzymes. 16 forms of -amylase of wheat & 5 forms of LDH (Lactate dehydrogenase) 3 Forms of Pepcase are known. These all isoenzyme forms are synthesised by different genes and tissue and organ specific.

Inducible enzymes - When formation of enzyme is induced by substrate availability. e.g. Lactase, Nitrogenase, -galactosidase.

Extremozymes - Enzymes, which may also function at extremely adverse conditions (very high temperature) e.g. Taq polymerase.

Abzymes - When the monoclonal antibodies (Mab) are used as enzymes.

Biodetergents - Enzymes used in washing powders are known as bio-detergents e. g.-amylase, lipase, proteolytic enzymes.

House keeping/constitutive enzymes - Which are always present in constant amount & are also essential to cell. Ex. Enzymes of cell respiration.

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Metal ion Metalloenzyme

Fe++, Fe+++ Cytochrome oxidase, catalase, aconitase, peroxidase

Ca++ Lipase, Succinic dehydrogenase, thrombin, thrombokinase

Mg++ Hexokinase, pyruvate kinase, DNA Polymerase, enolase, phosphotransferase

Cu++ Cytochrome oxidase, tyrosinase

Co++ Ascorbic acid oxidase, Peptidases

Mo Dinitrogenase, nitrate reductase

Mn++ Ribouncieotide reductase, Arginase

Zn++ Alcohol dehydrogenase, Carbonic anydrase, LDH, carboxypeptidase,

Glycine reductase, thiolase

Se Glycine reductase, thiolase

K+ Pyruvate kinase

Ni Urease

Cl Salivary amylase

Na+ ATPase

Classification of Enzymes

Enzymes were variously named in the past.

Enzyme names such as ptyalin (salivary amylase), pepsin and trypsin, give no indication of their action.

Other enzymes such as amylase, sucrase, protease and lipase were named after the substrates on which they act-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 (belongs to the family Bromeliaceae).

Some like DNA polymerase indicate its specific action, polymerisation.

The Duclaux (1883) provided a system for naming enzymes by adding suffix -ase at the end of enzyme name.

In this system, each enzyme ends with an -ase and consists of two parts, the first part indicates its substrate and the second the reaction catalysed.

For example, glutamate pyruvate transaminase transfers an amino group from the substrate glutamate to another substrate pyruvate.

However, arbitrary names like ptyalin and trypsin still continue to be used because of their familiarity.

Enzymes are grouped into six major classes:

Class 1. Oxidoreductases:

These catalyse oxidation or reduction of their substrates and act by removing or adding electrons (and/or H+) 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, lipases.

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 isomers, e.g., phosphohexose isomerase changes glucose-6-phosphate to fructose-6-phosphate (both are hexose phosphates).

Class 6. Ligases:

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

Factors Affecting Enzyme Activity

Tile activity of an enzyme can be affected by a change in the conditions which can alter the tertiary structure of the protein.

These include temperature, pH, change in substrate concentration or binding of specific chemicals that regulate its activity.

Temperature and pH

Enzymes generally function in a narrow range of temperature and pH (in figure).

Each enzyme shows its highest activity at a particular temperature and pH called the optimum temperature and optimum pH.

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 proteins are denatured by heat.

Effect of change in : (a) pH, (b) Temperature and

(c) Concentration of substrate on enzyme activity

(i) 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 called the optimum temperature.

Activity declines both above and below the optimum temperature.

Temperature:

Every enzyme has a specific optimum temperature.

According to the general rule of thumb the Q10 (temperature coefficient) for enzymes is 2-3, i.e., in between minimum and optimum temperature (5-40°C), the rate of reaction increases 2-3 times with rise in 10°C temperature.

If temperature is reduced to near or below freezing point, the enzymes are inactivated (not denatured).

Most enzymes show maximum activity in a temperature range of 25-40°C.

Enzymes are thermolabile i.e., are denatured at high temperature.

The loss of catalytic properties begins at 35ºC and is almost complete around 60°C.

However, dried enzyme extracts can endure temperature of 100°C-120°C or even higher.

That is why, dry seeds can endure higher temperature than germinating seeds.

Thermal stability is thus an important quality of some enzymes isolated from thermophilic organisms.

(ii) 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 8.0.

(iii) Concentration of Substrate:

With the increase in substrate concentration, the velocity of the enzymatic reaction rises at first.

The reaction ultimately reaches a maximum velocity (Vmax) which is not exceeded by any further rise in 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.

Characteristics of Enzymes

1. Proteinaceous nature: All enzymes are chemically made up of proteins (except ribozyme and ribonuclease-P). They, however may have additional inorganic or organic substances for their activity.

2. Amphoteric nature: The enzymes are capable of ionizing either as an acid or as a base depending upon the acidity of the external solution. Hence, their nature is amphoteric i.e., they can act as acid as well as base.

3. Colloidal nature: They are colloidal in nature due to which they present a large surface area for reaction to take place. They are hydrophilic and form hydrosol in the cell.

4. Reversibility: Like a true catalyst enzymes have been found to accelerate the chemical reaction in either direction i.e., forward and backward depending upon the availability of suitable energy requirement, pH, concentration of end products and availability ot reactants.

5. Molecular weight: Enzymatic proteins are substances of high molecular weight. Peroxidase, which is one of the smallest enzymes, has a molecular. weight of 40,000 whereas catalase, one of the largest enzymes, has a molecular weight of 250,000 (Urease 483,000).

6. Specificity of enzyme: Enzymes are highly specific in nature i.e. , a particular enzyme can catalyze only a particular type of reaction e.g., the enzyme malic dehydrogenase removes hydrogen atom from malic acid and not from other keto acids. The specificity of enzyme is determined by sequence of amino acids in the active sites. The active site possesses a particular binding site which complexes only with specific substrate. Thus a suitable substrate fulfils the requirements of active site and closely fixes with it.

7. Unchanged form: Enzymes are in no way transformed or used up in the chemical reaction but come out unchanged at the end of reaction.

8. Chemical reaction: Enzymes do not start a chemical reaction but increase the rate of chemical reaction. They do not change the equilibrium as well. However, they increase the rate of chemical reaction and bring about equilibrium very soon. Carbonic anhydrase is the fastest acting enzyme.

In absence of any enzyme this reaction is very slow, with about 200 molecules of H2C03 being formed in an hour. However, by using the enzyme present within the cytoplasm called carbonic anhydrase, the reaction speeds dramatically with about 600,000 molecules being formed every second. Hence, the enzyme accelerates the reaction rate by about 10 million times.

9. Efficiency: Efficiency of an enzyme is judged by its 'turn over number' i.e., number of substrate molecules changed per minute by a molecule of enzyme. It depends upon the number of active sites present over an enzyme, precise collisions between reactants and the rate of removal of end products The optimum turn-over number for enzyme carbonic anhydrase is 36 million, catalase 5 million, sucrase or invertase 10,000 and flavoprotein 50.

How Enzymes Speed Up Reactions?

A certain amount of energy is necessary to initiate any chemical reaction.

This is called activation energy or free energy of activation.

In a population of molecules of each substrate, the majority have average kinetic energy, some have higher and some lower than the average energy.

Under normal temperature, only the molecules having relatively high energy are likely to react to form the product.

Therefore, the reaction takes place very slowly.

One way to make the reaction go faster is to raise the temperature of the mixture.

Heat increases the kinetic energy of the molecules, causing their collisions and reaction.

The other method of quickening the reaction is by adding an enzyme.

The enzyme lowers the activation energy of the reaction and allows a large number of molecules to react at time.

Exactly how the enzymes lower the activation energy is not clear.

However, it is known that the enzymes combine with the substrate molecules and bring them close together which favours their collisions in the most suitable directions and locations for the reaction to occur.

The inorganic catalysts work in the same manner.

It is now held that conformational changes in the active sites of the enzymes actually "push" the substrate molecules toward an interaction.

Hydrolysis of starch into glucose is an organic chemical reaction.

Rate of a physical or chemical process refers to the amount of product formed per unit time.

Rate can also be called velocity if the direction is specified for a given reaction.

Activation energy requirement of uncatalysed and enzyme-catalysed reactions.

Reactants absorb energy from surroundings to climb the hill of activation energy (EA) and reach the unstable, short lived, transition state. Enzyme speeds up the reaction by reducing the uphill climb to the transition state. In transition state, the reactants are in an unstable condition and reaction can occur.

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1. Some laundry detergents contain enzymes. Generally proteases and amylases are added in detergents used for washing clothes and amylase in detergents used for dish washing. They help to break down proteins and other substances from food that may stain clothing.

2. Dairy digestive supplements contain lactase.

3. Baby foods: Trypsin is added to partially pre-digest the food.

4. Onions: Onions contain sulfur compounds called amino acid sulfoxides which result in the flow of lacrimal fluid.

Mode of Enzyme Action

1. Lock and key Hypothesis was put forward by Emil Fischer in 1894.

2. Induced Fit Theory was proposed by Koshland in 1959. According to this theory the active site of the enzyme contains two groups, buttressing and catalytic. The buttressing group is meant for supporting the substrate.

Mechanism of Enzyme Action

Two hypothesis have been put forward to explain the mode of enzyme action.

1. Lock and Key Hypothesis:

This hypothesis was given by Emil Fischer (1894).

According to this hypothesis, both enzyme and substrate molecules have specific geometrical shapes.

It is similar to the system of lock and key, which have special geometrical shapes in the region of their activity.

The active sites contain special groups having -NH2, -COOH, -SH for establishing contact with the substrate molecules.

Just as a lock can be opened by its specific key, a substrate molecule can be acted upon by a particular enzyme.

This also explains the specificity of enzyme action.

After coming in contact with the active site of the enzyme, the substrate molecules or reactants form a complex called enzymesubstrate complex.

In the enzyme substrate complex, the molecules of the substrate undergo chemical change and form products.

The product no longer fits into the active site and escapes in surrounding medium, leaving the active site free to receive more substrate molecules.

This theory explains how a small concentration of enzyme can act upon a large amount of the substrate.

It also explains how the enzyme remains unaffected at the end of chemical reaction.

The theory explains how a substance having a structure similar to the substrate can work as competitive inhibitor.

Lock and key hypothesis to show the specificity of enzymes

Enzyme + Substrate Enzyme — Substrate Complex

Enzyme — Substrate Complex Enzyme + End Products

2. Induced Fit Hypothesis:

This hypothesis was proposed by Koshland (1960).

According to this hypothesis the active site of the enzyme does not initially exist in a shape that is complementary to the substrate but is induced to assume the complementary shape as the substrate becomes bound to the enzyme.

According to Koshland, "the active site is induced to assume a complementary shape in much the same way as a hand induces a change in the shape of a glove."

An active site of an enzyme is a crevice or a pocket into which the substrate fits.

Thus, enzymes through their active site, catalyse reactions at a high rate.

Hence, according to this model, the enzyme (or its active site) is flexible.

Induced-fit theory of enzyme action. A. active site of enzyme. B. substrate molecule.

C. Enzyme-substrate complex with conformational changes so as to bring the

catalytic group against the substrate bonds to be broken

The active site of the enzyme contains two groups-

(a) Buttressing group is meant for supporting the substrate.

(b) Catalytic group is meant for catalysing the reaction.

When substrate comes in contact with the buttressing group, the active site changes to bring the catalytic group opposite the substrate bonds to be broken.

Iso-enzymes

Multiple molecular forms of an enzyme (synthesized by different genes) occurring in the same organism and having a similar substrate activity. Over 100 enzymes are known to have iso-enzymes such as

(i) -amylase of wheat endosperm has 16-iso-enzymes.

(ii) Lactic acid dehydrogenase has 5 iso-enzymes.

(iii) Alcohol dehydrogenase has 4 iso-enzymes.

Site of Enzyme Action

All enzymes are produced in the living cells.

About 2,000 enzymes have been recorded.

These are of two types with regard to the site where they act: intracellular and extracellular.

1. Intracellular Enzymes:

Most of the enzymes remain and function inside the cells.

They are called the intracellular enzymes or endoenzymes.

Some are dissolved in the cytoplasmic matrix.

A water extract of ground up liver cells contains all the eleven enzymes necessary to change glucose to lactic aid.

Certain enzymes are bound to particles, such as ribosomes, mitochondria and chloroplast.

The respiratory enzymes needed to convert lactic acid to carbon dioxide and water are found in the mitochondria.

2. Extraceliular Enzymes:

Certain enzymes leave the cells and function outside them.

They are called the extracellular enzymes or exoenzymes.

They mainly include the digestive enzymes, e.g., salivary amylase, gastric pepsin, pancreatic lipase; secreted by the cells of the salivary glands, gastric glands and pancreas, respectively.

Lysozyme present in tears and nasal secretion is also an exoenzyme.

The enzymes retain their catalytic action even after extraction from the cells.

Rennet tablets, containing the enzyme rennin from the calf's stomach, are used to coagulate milk protein caseinogen for cheese (casein) formation.

Inhibition of Enzyme Action

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 enzyme activity, the process is called inhibition and the chemical is called an inhibitor.

Following types of enzyme inhibition can occur :

(i) 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 inhibitor is called a Competitive Inhibitor of that enzyme.

Due to its close structural similarity with the substrate, the inhibitor competes with the latter for the substratebinding site of the enzyme.

Consequently, the enzyme cannot participate in catalysing the change of the substrate.

As a result the enzyme action declines, e.g., the inhibition of succinic 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.

Competitive inhibition of enzyme action

(ii) 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 (cytochrome c) 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.

(iii) Allosteric Modulation or 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(s) 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.

Examples :

(a) Hexokinase which changes glucose to glucose-6-phosphate in glycolysis. Decline in enzyme activity by the allosteric effect of the product is called Feedback Inhibition, e.g., allosteric inhibition of hexokinase by glucose-6-phosphate.

(b) Enzyme phosphofructokinase is activated by ADP and inhibited by ATP.

(c) Another example is inhibition of threonine deaminase by isoleucine. Amino acid isoleucine is formed in bacterium Escherichia coli in a 5-step reaction from threonine. When isoleucine accumulates beyond a threshold value, its further production stops.

 

Inhibition of Enzyme Activity

Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called an inhibitor.

Reversible inhibitors bind to enzymes through non-covalent bonds.

Dilution of the enzyme-inhibitor complex results in dissociation of the reversibly-bound inhibitor and recovery of enzyme activity.

Irreversible inhibition occurs when an inhibited enzyme does not regain activity upon dilution of the enzyme-inhibitor complex.

Some irreversible inhibitors act by forming covalent bonds with specific groups of enzymes; for example, the neurotoxic effects of certain insecticides are due to their irreversible binding at the catalytic site of the enzyme acetylcholinesterase.

The two most commonly encountered types of inhibition are competitive and noncompetitive.

A. Competitive inhibition :

This type of inhibition occurs when the inhibitor binds reversibly to the same site that the substrate would normally occupy, therefore, competes with the substrate for that site.

1. Effect on Vmax: The effect of a competitive inhibitor is reversed by increasing [S]. At a sufficiently high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of inhibitor.

2. Effect on Km : A competitive inhibitor increases the apparent Km for a given substrate. This means that in the presence of a competitive inhibitor more substrate is needed to achieve 1/2 Vmax, e.g., sulpha drugs for folic acid synthesis in bacteria and inhibition of succinic dehydrogenase by Malonate.

Effect of a competitive inhibitor on the reaction

velocity (v0) versus substrate [S] plot.

B. Non-competitive inhibition:

This type of inhibition is recognized by its characteristic effect on Vmax. Noncompetitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme. The noncompetitive inhibitor can bind either free enzyme or the ES complex, thereby preventing the reaction from occurring.

1. Effect on Vmax : Noncompetitive inhibition cannot be overcome by increasing the concentration of substrate. Thus, noncompetitive inhibitors decrease the Vmax of the reaction.

2. Effect on Km : Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme. Thus, the enzyme shows the same Km in the presence or absence of the noncompetitive inhibitor. e.g., cyanide kills an animal by inhibiting cytochrome oxidase.

Effect of a noncompetitive inhibitor on the reaction

velocity (v0) versus substrate [S] plot.

Summary

Although there is a bewildering diversity of living organisms, their chemical composition and metabolic reactions appear to be remarkably similar.

The elemental composition of living tissues and non-living matter appears to be similar when analysed qualitatively.

However, a closer examination reveals that the relative abundance of carbon, hydrogen and oxygen is higher in living systems when compared to inanimate matter.

The most abundant chemical in living organisms is water.

There are thousands of small molecular weight (< 1000Da) biomolecules.

Amino acids, monosaccharide and disaccharide sugars, fatty acids, glycerol, nucleotides, nucleosides and nitrogen bases are some of the organic compounds present in living organisms.

There are 21 types of amino acids and 5 types of nucleotides.

Fats and oils are glycerides in which fatty acids are esterified to glycerol.

Phospholipids contain, in addition, a phosphorylated nitrogenous compound.

They are found in cell membrane.

Lecithin is one example of a phospholipid.

Living organisms have a number of carbon compounds in which heterocyclic rings can be found.

Some of these are nitrogenous bases -Adenine, guanine, cytosine, uracil and thymine.

When found attached to a sugar, they are called nucleosides.

If a phosphate group is also found esterified to the sugar they are called nucleotides.

Adenosine, guanosine, thymidine, uridine and cytidine are nucleosides.

Adenylic acid, thymidylic acid, guanylic acid, uridylic acid and cytidylic acid are nucleotides.

Only three types of macromolecules, i.e., proteins, nucleic acids and polysaccharides are found in living systems.

Lipids, because of their association with membranes separate in the macromolecular fraction. Biomacromolecules are polymers.

They are made of building blocks which are different.

Proteins are heteropolymers made of amino acids. Nucleic acids (RNA and DNA) are composed of nucleotides.

Biomacromolecules have a hierarchy of structures primary, secondary, tertiary and quaternary.

Nucleic acids serve as genetic material.

Polysaccharides are components of cell wall in plants, fungi and also of the exoskeleton of arthropods.

They also are storageforms of energy (e.g., starch and glycogen).

Proteins serve a variety of cellular functions.

Many of them are enzymes, some are antibodies, some are receptors, some are hormones and some others are structural proteins.

Collagen is the most abundant protein in animal world and Ribulose bisphosphate Carboxylase-Oxygenase (RuBisCO) is the most abundant protein in the whole of the biosphere.

Enzymes are proteins which catalyse biochemical reactions in the cells.

Ribozymes are nucleic acids with catalytic power.

Proteinaceous enzymes exhibit substrate specificity, require optimum temperature and pH for maximal activity.

They are denatured at high temperatures.

Enzymes lower activation energy of reactions and enhance the rate of the reactions greatly.

Nucleic acids carry hereditary information and are passed on from parental generation to progeny.

In some cases non-protein constituents called cofactors are bound to the enzyme to make the enzyme catalytically active.

In these instances, the protein portion of the enzymes is called the apoenzyme.

Three kinds of cofactors may be identified; prosthetic groups, coenzymes and metal ions.

Prosthetic groups are organic compounds and are distinguished from other cofactors in that they are tightly bound to the apoenzyme.

For examples, in peroxidase and catalase, which catalyse the breakdown of hydrogen peroxide to water and oxygen, haem is the prosthetic group and it is a part of the active site of the enzyme.

Co-enzymes are also organic compounds but their association with apoenzyme is only transient, usually occurring during the course of catalysis.

NAD, NADP are co-enzymes and contain niacin vitamin.

A number of enzymes require metal ions for their activity which form coordination bonds with side chains at the active site e.g. zinc is a cofactor for the proteolytic enzyme carboxypeptidase.

Enzymes

Proteins make up almost all enzymes. Some nucleic acids have enzyme-like properties. Ribozymes are the name for these proteins. A line diagram can be used to represent an enzyme. An enzyme, like any other protein, has a primary structure, which is the amino acid sequence. Secondary and tertiary structures are present in enzymes, as they are in other proteins. When you look at a tertiary structure, you'll observe that the protein chain's backbone folds in on itself, the chain crisscrosses itself, and many crevices or pockets form. The 'active site' is one such pocket. An enzyme's active site is a crevice or pocket that the substrate fits into. As a result, enzymes catalyze processes at a rapid pace through their active site.

In many aspects, enzyme catalysts differ from inorganic catalysts, but one key distinction stands out. At high temperatures and pressures, inorganic catalysts perform efficiently, but enzymes are destroyed at high temperatures (say, above 40°C). Enzymes extracted from organisms that dwell in severe heat (e.g., hot vents and sulphur springs) are stable and keep their catalytic power even at extreme temperatures (up to 80°-90°C). The capacity of such enzymes extracted from thermophilic organisms to maintain their thermal stability is thus critical.

Chemical reactions

Chemical compounds go through two sorts of transformations. A physical change is merely a change in shape that does not involve the breaking of connections.This is a bodily function. A change in state of matter is another physical process, such as when ice melts into water or when water becomes vapour. These are physical processes as well. A chemical reaction, on the other hand, occurs when bonds are broken and new bonds are generated during transformation. For example

Ba (OH)2+ H2SO4 ® BaSO4 + 2H2O

is a chemical process that takes place in an inorganic state. The conversion of starch to glucose is also an organic chemical reaction. The amount of product created per unit time is referred to as the rate of a physical or chemical process. It can be written as:

rate=dP/ dT

If the direction is indicated, rate is also known as velocity. Temperature, among other things, affects the rates of physical and chemical processes. For every 10°C change in either direction, the rate doubles or declines by half, according to a common rule of thumb. Catalyzed reactions proceed at a much faster rate than uncatalyzed reactions. For every 10°C change in either direction, the rate of enzyme catalysed reactions would be substantially larger than the same but uncatalyzed reaction. Catalyzed reactions proceed at a much faster rate than uncatalyzed reactions. When enzyme-catalyzed reactions are detected, the rate is much higher than when the identical process is not catalysed. As an example,

In the absence of any enzyme, this reaction is extremely sluggish, producing just around 200 molecules of H2CO3 per hour. The reaction speeds up substantially when carbonic anhydrase, an enzyme found in the cytoplasm, is used, with roughly 600,000 molecules generated every second. The enzyme has sped up the reaction rate by a factor of ten million. The power of enzymes is very amazing! Thousands of different enzymes exist, each catalysing a different chemical or metabolic reaction. A metabolic route is a multistep chemical reaction in which each step is catalysed by the same enzyme complex or separate enzymes. As an example,

is a metabolic pathway in which glucose is converted to pyruvic acid via 10 enzyme-catalyzed chemical events. At this point, you should be aware that this metabolic pathway, when supplemented with one or two additional processes, produces a wide range of metabolic end products. Lactic acid is produced in skeletal muscle under anaerobic circumstances. Pyruvic acid is generated under typical aerobic circumstances. The similar route leads to the generation of ethanol in yeast during fermentation (alcohol). As a result, different products are possible depending on the circumstances.

How do Enzymes bring about such High Rates of Chemical Conversions?

The concept of an active site is already known. A reaction is referred to as a chemical or metabolic change. A'substrate' is the chemical that is transformed into a product. As a result, enzymes, which are three-dimensional proteins with a 'active site,' convert a substrate (S) into a product (P). This can be represented symbolically as

Within a specific cleft or pocket, the substrate 'S' must bind the enzyme at its'active site.' The substrate must diffuse into the 'active site.' As a result, the creation of a 'ES' complex is required. The letter E stands for enzyme. This intricate structure is a one-time occurrence. A new structure of the substrate called the transition state structure is created when the substrate is attached to the enzyme active site. The product is quickly removed from the active site after the intended bond breaking/making is completed. In other words, the substrate's structure is changed into the product's structure (s). This transformation must pass via a structure known as a transition state structure.Between the stable substrate and the result, there could be many additional 'altered structural states.' The notion that all other intermediate structural states are unstable is implicit in this assertion. Stability has to do with the molecule's or structure's energy status.

Figure 8: Concept of energy activation

The potential energy content is shown by the y-axis. The x-axis depicts the structural transformation or states as they proceed through the 'transition stage.' There would be two things that you would notice. The difference in energy levels between S and P. When 'P' is less than 'S,' the reaction is exothermic. It is not necessary to provide energy (through heating) in order to manufacture the product. Regardless of whether the reaction is exothermic or spontaneous or endothermic or energy-demanding, the 'S' must pass through a significantly higher energy level or transition state. 'Activation energy' is the difference between the average energy content of 'S' and that of this transition state. Enzymes gradually break down this energy barrier, allowing the 'S' to become 'P' more easily.

Nature of Enzyme Action

In order to create a highly reactive enzyme-substrate complex (ES), each enzyme (E) has a substrate (S) binding site in its molecule. This complex has a brief half-life and dissociates into its product(s) P and the unmodified enzyme, with the enzyme-product complex forming in the middle (EP). Catalysis requires the creation of the ES complex.

E+S → ES → EP → E+P

An enzyme's catalytic cycle can be broken down into the following steps:

1. The substrate first binds to the enzyme's active site, fitting into the active site.

2. The enzyme's shape changes as a result of the substrate's binding, causing it to fit more tightly around the substrate.

3. The enzyme's active site, which is now in close proximity to the substrate, breaks the substrate's chemical bonds, forming a new enzyme-product complex.

4. The reaction products are released by the enzyme, and the free enzyme is ready to bind to another molecule of the substrate and repeat the catalytic cycle.

Factors Affecting Enzyme Activity

A change in the circumstances can impact the activity of an enzyme by altering the protein's tertiary structure. Temperature, pH, changes in substrate concentration, and the binding of certain molecules that affect its activity are examples of these.

Temperature and pH: Enzymes typically operate within a narrow temperature and pH range. The optimum temperature and pH for each enzyme are defined as the temperature and pH at which the enzyme is most active. Both below and above the ideal value, activity decreases. Because proteins are denatured by heat, low temperatures maintain the enzyme in a temporarily inactive state, but high temperatures eliminate enzymatic function.

  Figure 9 Factors affecting enzyme activity

Substrate Concentration: At first, the enzymatic reaction's velocity increases as the substrate concentration rises. The reaction eventually achieves a maximum velocity (Vmax) that isn't exceeded by any further increase in substrate concentration. This is because there are fewer enzyme molecules than substrate molecules, and there are no free enzyme molecules to interact with the additional substrate molecules after these molecules have been saturated. An enzyme's activity is also affected by the presence of certain molecules that bind to it. The process of inhibition occurs when a chemical binds to an enzyme, and the chemical is referred to as an inhibitor.Competitive inhibitors are those that have a chemical structure that closely mimics that of the substrate and inhibits the enzyme's activity. Because of its structural similarity to the substrate, the inhibitor competes with it for the enzyme's substrate binding site. As a result, the substrate is unable to attach, and the enzyme's activity is reduced, as seen in the inhibition of succinic dehydrogenase by malonate, which is structurally similar to the substrate succinate. Competitive inhibitors like these are frequently employed to combat bacterial infections.

Classification and Nomenclature of Enzymes

There have been thousands of enzymes identified, isolated, and analyzed. The majority of these enzymes have been divided into groups depending on the kind of reactions that they catalyze. Enzymes are classified into six classes, each having four to thirteen subclasses, and given a four-digit number.

Figure10: Enzyme classification

Co-factors

Enzymes are polypeptide chains made up of one or more polypeptide chains. However, in other situations, non-protein elements known as cofactors are coupled to the enzyme in order for it to be catalytically active. The apoenzyme refers to the protein part of the enzyme in these cases. Prosthetic groups, co-enzymes, and metal ions are all examples of cofactors. Prosthetic groups are chemical molecules that are strongly attached to the apoenzyme, distinguishing them from other cofactors. The prosthetic group haem is a component of the active site of enzymes like peroxidase and catalase, which catalyze the breakdown of hydrogen peroxide to water and oxygen.Co-enzymes are organic substances as well, although their relationship with the apoenzyme is very temporary, occurring most often during catalysis. Co-enzymes are also used as co-factors in a variety of enzyme-catalyzed processes. Many coenzymes contain vitamins as important chemical components; for example, the coenzymes nicotinamide adenine dinucleotide (NAD) and NADP contain the vitamin niacin. Metal ions that create coordination bonds with side chains in the active site while also forming one or more coordination bonds with the substrate are required for the activity of a number of enzymes, for example, zinc is a cofactor for the proteolytic enzyme carboxypeptidase. When a co-factor is removed from an enzyme, its catalytic activity is lost, indicating that they play an important part in the enzyme's catalytic activity.