Enzymes

History of cellular enzymes

Enzymes (Gk. en = in zyme = yeast) are proteinaceous substances which are capable of catalysing chemical reactions of biological origins without themselves undergoing any change. Enzymes are biocatalysts. An enzyme may be defined as 𠇊 protein that enhances the rate of biochemical reactions but does not affect the nature of final product”. Like the catalyst the enzymes regulate the speed and specificity of a reaction, but unlike the catalyst they are produced by living cells only. All components of cell including cell wall and cell membrane have enzymes.

Maximum enzymes (70%) in the cell are found in mitochondrion. Enzymes are also called ‘biological middle man’. The study of the composition and function of the enzyme is known as enzymology.

The term enzyme (meaning in yeast) was used by Willy Kuhne (1878) while working on fermentation. At that time living cells of yeast were thought to be essential for fermentation of sugar. Edward Buchner (1897), a German chemist proved that extract zymase, obtained from yeast cells, has the power of fermenting sugar (alcoholic fermentation). Zymase is complex of enzymes (Buchner isolated enzyme for the first time).

Later J.B. Sumner (1926) prepared a pure crystalline form of urease enzyme from Jack Bean (Canavalia ensiformis) and suggested that enzymes are proteins. Northrop and Kunitz prepared crystals of pepsin, trypsin and chymotrypsin Arber and Nathans got noble prize in 1978 for the discovery of restriction endonucleases which break both strands of DNA at specific sites and produce sticky ends. These enzymes are used as microscissors in genetic engineering.

Nature of enzymes

Mostly enzymes are proteinaceous in nature. With some exception all enzymes are proteins but all proteins are not enzymes. Enzymatic protein consist of 20 amino acids. The polypeptide chain or chains of an enzyme show tertiary structure. Their tertiary structure is very specific and important for their biological activity. Loss of tertiary structure renders the enzymic activity.

Some enzymes like pepsin, amylase, urease, etc., are exclusively made up of protein i.e., simple proteins. But most of the other enzymes have a protein and a non-protein component, both of which are essential for enzyme activity. The protein component of such enzymes is known as apoenzyme whereas the non-protein component is called cofactor or prosthetic group. The apoenzyme and prosthetic group together form a complete enzyme called holoenzyme.

Activity of enzyme is due to co-factor, which can be separated by dialysis. co-factor is small, heat stable and may be organic or inorganic in nature.

Three types of cofactors may be identified. Prosthetic group, coenzyme and metal ions.

Prosthetic group : Prosthetic groups are organic compounds distinguished from other cofactors in that they are permanently bound to the apoenzyme, e.g., in peroxisomal enzymes peroxidase and catalase which catalyzes breakdown of hydrogen peroxide to water and oxygen.

Coenzymes : Fritz Lipmann discovered coenzymes. Coenzymes are also organic compounds but their association with the apoenzyme is transient, usually occurring only during the course of catalysis.

In general coenzymes not only assist enzymes in the cleavage of the substrate but also serve as temporary acceptor for one of the product of the reaction. The essential chemical component of many coenzymes are vitamins, e.g., coenzyme nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP) contains the vitamin niacin, coenzyme A contains pantothenic acid, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) contains riboflavin (Vitamin B₂), and thiamine pyrophosphate (TPP) contains thiamine (Vitamin B­₁).

Metal ions : A number of enzymes require metal ions for their activity. The metal ions form coordination bonds with specific side chains at the active site and at the same time form one or more coordination bonds with the substrate. The latter assist in the polarization of substrate bonds to be cleaved by the enzyme. The common metal ions are Zn⁺⁺, Cu⁺⁺, Mg⁺⁺.

Inorganic part of enzyme acts as prosthetic group in few enzymes they are called activators. These activators are generally metals. Hence these enzymes are called Metalloenzyme such as :

Nomenclature and Classification

Dauclax, (1883) introduced the nomenclature of enzyme. Usually enzyme names end in suffix-ase to the name of substrate e.g., Lactase acts on lactose, maltase act on maltose, amylase on amylose, sucrase on sucrose, protease on proteins, lipase on lipids and cellulase on cellulose. Sometimes arbitrary names are also popular e.g., Pepsin, Trypsin and Ptylin etc. Few names have been assigned as the basis of the source from which they are extracted e.g., Papain from papaya, bromelain from pineapple (family Bromeliaceae). Enzymes can also be named by adding suffix-ase to the nature of chemical reaction also e.g., Oxidase, dehydrogenase, catalase, DNA polymerase.

Modern names are given after chemical action. They are more systamatic, informative but slightly longer. e.g., ATP : D-glucose phosphotransferase.

Common simpler names used at the place of systematic names called trivial names.

According to older classification : The older classification of enzymes is based on the basis of reactions which they catalyse. Many earlier authors have classified enzymes into two groups :

(1) Hydrolysing enzyme : The hydrolysing enzymes of hydrolases catalyse reactions in which complex organic compounds are broken into simpler compounds with the addition of water. Depending upon the substrate hydrolysing enzymes are :

Carbohydrases : Most of the polysaccharides, disaccharides or small oligosaccharides are hydrolysed to simpler compounds, e.g., hexoses or pentoses under the influence of these enzymes.

Lactase on lactose to form glucose to galactose, sucrase/invertase on sucrose to form glucose and fructose, amylase or diastase on starch to form maltose, maltase on maltose to form glucose, cellulase on cellulose to produce glucose.

Easterases : These enzymes catalyse the hydrolysis of substances containing easter linkage, e.g., fat, pectin, etc. into an alcoholic and an acidic compound.

$Fat\stackrel{Lipase}{\to }Glycerol+Fattyacid$

$Phosphoricacideasters\stackrel{Phosphatase}{\to }Phosphoricacid+Othercompounds$

Proteolytic enzymes : The hydrolysis of proteins into peptones, polypeptides and amino acids is catalysed by these enzyme

$Protein\stackrel{Pepsin}{\to }Peptones$

$Polypeptides\stackrel{Peptidases}{\to }Aminoacids$

Amidases : They hydrolyse amides into ammonia and acids.

$Urea\stackrel{Urease}{\to }Ammonia+Carbondioside$

$Asparagine\stackrel{Asparaginase}{\to }Ammonia+Asparticacid$

(2) Desmolysing enzymes :  Most of the desmolysing enzymes are the enzymes of respiration e.g., oxidases, dehydrogenases, (concerned with transfer of electrons), transaminases carboxylases etc.

According to IUB system to classification : In 1961 the Commission on enzymes set up by the ‘International Union of Biochemistry’ (IUB) framed certain rules of their nomenclature and classification.

According to IUB system of classification the major points are :

$\bullet$ Reactions (and enzymes catalyzing them) are divided into 6 major classes each with 4-13 subclasses.

$\bullet$ The enzyme name has two parts-first name is of substrate. The second ending in ase indicates type of reaction.

$\bullet$ The enzyme has a systematic code No. (Enzyme code/Enzyme Commission). The first digit denotes the class, the second sub-class, the third sub-sub-class and the fourth one is for the particular enzyme name. Thus, E.C. 2.7.1.1 denotes class 2 (Transferases)-subclass 7 (transfer of phosphate) sub-sub-class 1 (an alcohol functions as phosphate acceptor). The 4^th digit indicates hexokinase. Major classes of enzymes are as follows :

(i) Oxidoreductases : These enzymes catalyse oxidation reduction reactions, usually involving the transfer of hydrogen atoms or ions from one molecule to another. There are three main types of these enzymes :

Oxidases : Where the hydrogen is transferred from a molecule to oxygen, e.g., cytochrome oxidase. They play very important role in E.T.S. in photosynthesis as well as respiration,

Dehydrogenases : Where the hydrogen is transferred to a coenzyme such as NAD⁺, e.g., Succinic dehydrogenase. They help in oxidation of organic molecules during aerobic respiration.

Reductase :  It is cause addition of hydrogen or an electron and remove oxygen. e.g., Nitrate reductase requires NAD (coenzyme I) as coenzyme for the reaction.

(ii) Transferases : These enzyme catalyse the transfer of a specific group (e.g., amino, methyl, acyl, phosphate) from one kind of molecule to another e.g., transphosphorylases, transaminases, transpeptidases, transmethylases, kinases, etc.

(iii) Hydrolases : These enzyme catalyse the hydrolysis of organic foods i.e., the breakdown of large molecules by addition of water. Most of the hydrolysing (digestive) enzymes are located in lysosomes. e.g., all digestive enzymes such as lipases (digest the stored food material of castor seeds) amylases, esterases, phosphatases, carbohydrases, proteases.

(iv) Lyases : These enzymes catalyse the breakage of specific covalent bonds and removal of groups without hydrolysis e.g., fumerases, carboxylases, aminases, histidine decarboxylase that splits C-C-bond of histidine, forming CO₂ and histamine.

(v) Isomerases : These enzymes catalyse the rearrangement molecular structure to form isomers. e.g., phosphohexose isomerase (phosphoglucomutase) act on glucose 6-phosphate to form fructose 6-phosphate (both C­₆ compounds) epimerase, racemase.

(vi) Ligases or Synthetases : These enzymes form bondsਊnd join two molecules together, using energy supplied from the breakdown of ATP, e.g., DNA ligase is used to repair breaks in DNA molecules. Amino-Acyl synthetase is used to activate t-RNA by attaching amino acid at 3¢ end. Tryptophan synthetase is used to convert tryptophan amino acid to IAA, etc.

Site of enzyme action

All enzymes are produced in the living cells. About 3,000 enzymes have recorded. These are of two types with regard to the site where they act as :

Intracellular enzymes : Most of the enzymes remain and function inside the cells, They are called the intracellular enzymes or endoenzymes. Some of these enzymes are found in cytoplasmic matrix. Certain enzymes are bound to ribosomes, mitochondria and chloroplast etc.

Extracellular 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, lysozyme present in tears and nasal secretion.

Rennet tablets with enzyme renin from calf’s stomach are widely used to coagulate protein caseinogen for cheese (casein) formation.

Mechanism of enzyme action

Energy is required to bring the inert molecules into the activated state. The amount of energy required to raise the energy of molecules at which chemical reaction can occur is called activation energy. Enzymes act by decreasing the activation energy so that the number of activated molecules is increased at lower energy levels. If the activation energy required for the formation of the enzyme-substrate complex is low, many more molecules can participate in the reaction than would be the case if the enzyme were absent.

Mode of enzyme action

In 1913 Michaelis and Menten proposed that for a catalylic reaction to occur it is necessary that enzyme and substrate bind together to form an enzyme substrate complex.

\mathop {\rm{E}}\limits_{{\rm{(Enzyme)}}}#xA0+ \mathop {\rm{S}}\limits_{({\rm{Substrate)}}}#xA0\to \mathop {{\rm{E - S Complex}}}\limits_{({\rm{Enzyme - substrate Complex)}}}

{\rm{E - S Complex}} \to \mathop {\rm{E}}\limits_{{\rm{(Enzyme)}}}#xA0+ \mathop {\rm{P}}\limits_{({\rm{Product)}}}

It is amazing that the enzyme-substrate complex breaks up into chemical products different from those, which participated in its formation (i.e., substrates). On the surface of each enzyme there are many specific sites for binding substrate molecules called active sites or catalytic sites.

There are two views regarding the mode of enzyme action :

Lock and Key hypothesis : The hypothesis was put forward by Emil Fisher (1894). According to this hypothesis the enzyme and its substrate have a complementary shape. The specific substrate molecules are bound to a specific site of the enzyme molecule.

The theory can be explained easily by the fact that a particular lock can be opened by a particular key specially designed to open it. Similarly enzymes have specific sites where a particular substrate can only be attached. The lock and key model accounts for enzyme specificity.

Induced fit hypothesis : This hypothesis was proposed by Daniel, E. Koshland (1959).

According to this view, active site is not rigid but static and it has two groups ­- buttressing group and catalytic group. Initially substrate bind to the buttressing group which induces the catalytic group to fit the substrate and catalytic group weakes the bonds of reactant or substrate by electrophilic and nucleophilic forces.

Properties of enzymes

Molecular weight : Enzymatic proteins are substances of high molecular weight. Bacterial ferredoxin one of the smaller enzymes has molecular weight of 6,000, where as pyruvic dehydrogenase one of the largest-has a molecular weight of 4600000.

Amphoteric nature : Each molecule of enzyme possess numerous groups which yield H⁺ in slightly alkaline solutions and groups which yield OH^- ions in slightly acidic solutions. Unlike many other substances, therefore, the enzymatic protein is amphoteric, i.e., capable of ionizing either as an acid or as a base depending upon the acidity of the external solution.

Colloidal nature : All enzymes are colloidal in natureਊnd thus provide large surface area for reaction to take place. They posses extremely low rates of diffusion and form colloidal system in water.

Specificity of enzyme : Most of the enzymes are highly specific in their action. A single enzyme will generally catalyze only a single substrate or a group of closely related substrates. The active site possess a particular binding site which complexes only with specific substrate. Thus, only a suitable substrate fulfils the requirements of active site and closely fixes with it.

Heat specificity : The enzymes are thermolabile i.e., heat sensitive. They function best at an optimum temperature (20°C-40°C). Their activity decrease with decrease as well as increase in temperature and stops at 0ଌ and above 80°C.

Catalytic properties : Enzymes are active in extremely small amounts, e.g., one molecule of invertase can effectively hydrolyze 1,000,000 times its own weight of sucrose. One molecule of catalase is able to catalyze conversion of 5,000,000 molecules of hydrogen peroxide.

Reversibility of reaction : The enzyme-controlled reactions are reversible. The enzymes affect only the rate of biochemical reactions, not the direction. e.g., Lipase can catalyse splitting of fat into fatty acids and glycerol as well as synthesis of fatty acids and glycerol into fats.

pH sensitivity : The enzymes show maximum activity at an optimum pH is 6-7.05(7 $\pm$ 1.05). Their activity slows with decrease and increase in pH till it stops. Each enzyme has its own different favourable pH value.

High efficiency : The effectiveness of an enzymatic reaction is expressed in terms of its turn over number or catalytic centre activity means number of substrate molecules on which one enzymes molecules acts in one minute.

Turn over number depends on the number of active sites of an enzyme. An active site is an area of the enzyme which is capable of attracting and holding particular substrate molecules by its specific charge, size and shape so as to allow the chemical change, Enzymes show 3-D structure. R (alkyl) groups of amino acids from active sites during folding polypeptide chains. Usually 3-12 amino acids form an active site.

Highest turn over number is of carbonic anhydrase (36 million/min or 600000 per second) and lowest is of lysozymes (30/min or 0.5 per second). So carbonic anhydrase is fastest enzyme. The lowest turn over number is of lysozymes.

Enzyme inhibition

Competitive inhibition : Substances (inhibitors) which are structurally similar to the substrates and competes for the active site of the enzyme are known as competitive inhibitors. Usually such inhibitors show a close structural resemblance to the substrates to the enzyme they inhibit. In such a case, inspite of enzyme substrate complex, enzyme inhibitor complex is formed and enzyme activity is inhibited.

\mathop {\rm{E}}\limits_{{\rm{Enzyme}}}#xA0+ \mathop {\rm{I}}\limits_{{\rm{inhibitor}}}#xA0\to \mathop {{\rm{EI}}}\limits_{{\rm{Enzyme}} - {\rm{inhibitor}}\,{\rm{complex(EI)}}}

The concentration of EI complex depends on the concentration of free inhibitor. Because EI complex readily dissociates, the empty active sites are then available for substrate binding. The effect of a competitive inhibitor on activity is reversed by increasing the concentration of substrate. In it V_max remain constant and Km increases.

A classic example of competitive inhibition is succinic acid dehydrogenase which oxidises succinic acid to fumaric acid. If concentration of malonic acid, is added, the activity of succinic dehydrogenase decreases rapidly. Hence malonic acid acts as a competitive inhibitor since it has structural resemblance to succinc acid.

The competitive inhibition can be reversed by increasing the concentration of the substrate. Competitive inhibitors are used in control of bacterial pathogens.

Non-competitive inhibition : These substances (poisons) do not combine with active sites but attach somewhere else and destroy the activity of enzyme.

Both EI and ES complexes are formed. Inhibitor binding alters the three dimensional configuration of the enzyme and thus blocks the reaction. Non competitive inhibitor do not competes directly with the substrate for binding to the enzyme. In it V_max in lowered and Km is changed.

The non-competitive inhibition can not be reversed by increasing the concentration of the substrate i.e., irreversible. e.g., cyanide inhibits the mitochondrial enzyme cytochrome oxidase which is essential for cellular respiration. This kills the animals.

More AMP is a non competitive inhibitor of fructose biphosphate phosphatase, the enzyme that catalyzes the conversion of fructose 1, 6 biphosphate to fructose 6 phosphate.

Feedback inhibition : In number of cases, accumulation of the final product of the reaction is capable of inhibiting the first step of reaction.

The product P checks the activity of enzyme which converts A into B. It is quite useful mechanism because it checks the accumulation of products.

The phenomenon in which the end product of a metabolic pathway can regulate its own production by inhibition of the sort is called feed back inhibition or negative feed back inhibition. This type of inhibition can be shown in Escherichia coli bacterium which synthesises the amino acid isoleucine from a substrate threonine by a series of intermediate reactions (i.e., $\alpha$ketobutyrate threonine deaminase, $\alpha$Aceto hydroxy butyrate, $\alpha$keto$\beta$methyl valerate etc).

When isoleucine accumulates in amounts more than required, it stops its own production by inhibiting the activity of the enzyme. Threonine deaminase which catalyzes the first reaction of the series. This type of metabolic control in which the first enzyme of a series is inhibited by the end product, is known as end product inhibition.

Allosteric inhibition (Modulation) : Allosteric literally means 𠆊nother place’. Still other inhibitors join an enzyme at a specific site and change the form of the active site meant for the substrate. These inhibitors are known as modifiers or modulators and the sites where they fit in are called allosteric sites. Modulators are of two types-positive (activators) and negative (inhibitors).

Change of active site form prevent the binding of substrate to the enzyme and stops the reaction. The process is called allostery or allosteric inhibition, The enzyme with allosteric sites are called allosteric enzymes. Jacob and Monod have termed this phenomenon as allosteric transition.

An example of allosteric enzyme inhibition is hexokinase that converts glucose to glucose 6-phosphate. Glucose 6-phosphate causes allosteric inhibition of hexokinase. This is called feedback allosteric inhibition.

Some terms regarding enzymes

Zymogens or (Enzyme Precursors) : Certain enzymes are produced by the living cells in an inactive (non-functional) form. They are called the zymogens or proenzymes. It is then converted, usually by proteolysis (hydrolysis of the protein), to the active form when it has reached the site of its activity. Pepsinogen and trypsinogen are zymogens produced by gastric glands and pancreas respectively. They are necessary to life because they degrade dietary proteins into amino acids that are used by the cell.

Pepsinogen is changed to active pepsin by hydrogen ions in the stomach. Trypsinogen is activated to trypsin by an enzyme enterokinase in the small intestine. Once small amount of pepsin or trypsin is formed, it itself catalyzes the activation of remaining proenzyme. This process is called autocatalytic reaction, or autocatalysis.

Isoenzymes (Isozymes) : There are certain enzymes which have slightly different molecular structure but performing the same catalytic function. Such enzymes are called isoenzymes or simply isozymes. Isoenzyme of an enzyme differ from each another in their amino acid sequence, molecular weight, immunological and electrophoretic behaviours. Hence, they can be separated by electrophoresis.

More than 100 enzymes are known to have isoenzyme. A good example of isoenzyme is lactic dehydrogenase (LDH). It catalyzes change of pyruvate to lactate. There are five LDH isoenzymes in muscles of heart. Alcohol dehydrogenase has four isoenzyme in maize. a-amylase (wheat endosperm) has 16 isoenzymes.

Inducible enzymes : An enzyme which is synthesized only in the presence of its substrate (inducer) is called inducible enzyme e.g., $\beta$-galactosidase.

Constitutive enzymes (House keeping enzyme) : The enzyme which are found in constant amounts under different growth conditions (regardless of its metabolic states) are called constitutive enzyme e.g., enzymes of sugar breakdown i.e., glycolysis.

Repressible enzymes : The presence of a specific substance may inhibit continued production of specific enzyme (enzyme repressor) e.g., glucokinase.

Ribozymes : Study of post transcriptional processing of RNA molecules has led to the most exciting discovery of the existence of some catalytic RNA molecules which have been called as RNA enzymes or ribozymes. All enzymes are not proteins as confirmed by Cech (1981) and Altman (1983). Ribozyme and RNAase-P are two non protein enzyme where RNA acts as catalyst. Ribozyme was reported from Tetrahymens (a protozoans) by Cech. The substrate for ribozyme is usually an RNA molecule. RNAase-P (Ribonuclease) was discovered by Altman.

Peptidyl transferase is also a non-proteinaceous enzyme, discovered by Noller.

Michaelis constant : Michaelis and Menten (1913) introduced a constant K_m (Michaelis constant).

It is a mathematical derivative or constant which indicates the substrate concentration at which the chemical reaction catalysed by an enzyme attains half its maximum velocity (V_max).

K_m indicates affinity of the enzyme for its substrate.

                              $K_m=\frac{1}{2}{V}_{\max }$

K_m value differs from substrate to substrate because different enzymes differ in their affinity towards different substrates. A high K_m indicates low affinity while a low K_m shows strong affinity. Protease acts on different proteins. So it’s K_m value will differ from protein to protein.

The Michaelis Menten equation description how reaction relatively varies with substrate concentration as given

V₀ =$\frac{V_{\max }[S]}{K_m+[S]}$

Where V₀ is the rate of initial reaction V_max is the maximum relative or the reaction rate with excess substrate K_m is the Michaelis constant =K₂+K₃/K₁ [S] is the substrate concentration.

The above reaction shows that the greater the affinity between an enzyme and its substrate, the lower the K_m (in units moles per litre) of the enzyme substrate reaction. Stated inversely, 1/K_m is the measure of affinity of the enzyme for its substrate.

Enzyme-inhibitor dissociation constant ­(Ki) : It is dissociation constant of enzyme - inhibitor complex.

Ki $=\frac{[\mathrm{E}][\mathrm{I}]}{[\mathrm{EI}]}$

Where, E is enzyme and I is concentration of inhibitor.

High Ki decreases enzyme activity while low Ki increases some, it is applicable to competitive inhibitors.

Factors affecting the enzyme activity

Substrate concentration : If there are more enzyme molecules than substrate molecules, a progressive increase in the substrate molecules increases the velocity of their conversion to products. However, eventually the rate of reaction reaches the maximum. At this stage the active sites of all the available enzyme molecules are occupied by the substrate molecules. Therefore, the substrate molecules occupy the active sites vacated by the products and cannot increase the rate of reaction further.

Enzyme concentration : The rate of reaction is directly proportional to enzyme concentration. An increase in enzyme concentration will cause a rise in the rate of reaction up to a point and them the rate of reaction will be constant. Increasing the enzyme concentration increases the number of available active sites.

Product concentration : Accumulation of the product of enzyme reaction lowers the enzyme activity. Enzyme molecules must be freed to combine with more substrate molecules. Normally the product are quickly removed from the site of formation and the reaction does not suffer.

Hydrogen ion concentration (pH) : Some enzyme act best in an acid medium, other in an alkline medium, for every enzyme there is an optimum pH where its action is maximum e.g., 2 for pepsin, 6.8 for salivary amylase, 8.5 for trypsin. Most enzyme show maximum activity in a pH range of about 6.0 to 7.5 i.e., near neutral pH (endoenzymes). A shift to the alkaline or acid side rapidly decreases the enzyme activity and finally stops it altogether. This is due to denaturation of enzyme molecule i.e., change in its physical structure.

Temperature : Within certain limits (5-40°C) the rate of an enzyme catalyzed reaction increases as the temperature increases. The Q₁₀ of most enzymatic reactions is 2, i.e., every 10°C rise in temperature doubles the rate of reaction. Most enzymes show maximum activity in a temperature range of 25 to 40°C. Beyond this temperature, there is sharp fall in the rate of reaction.

Modification in the physical form of the enzyme results in the loss of its catalytic activity. This change in structure is called denaturation of protein. This is the permanent change, and the denatured enzyme protein remains inactive even if the temperature is then brought down. The enzymes are not destroyed by freezing, and regain their lost activity if the temperature is raised to normal.

Deep freezing of food for preserving them for long periods is done not only to prevent the growth and multiplication of microorganisms but also to inactivate enzymes. It makes impossible for the microorganisms to digest the food.

Enzyme inhibitors : Certain chemical compounds inhibit activity of enzyme molecules either permanently or temporarily. Thus, di-isopropyl flurophosphate (DFP) inhibits the action of various enzymes catalysing hydrolysis of ester linkage. Inhibition is permanent or irreversible.

Poisons and Radiation : Poisons such as cyanide and radiation destroy the tertiary structure of the enzymes, making them ineffective.