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  • Cell Structure and Functions
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ENZYMES

  • Enzymes are commonly proteinaceous substances which are capable of catalyzing chemical reactions of biological origin without themselves undergoing any change. Therefore, they are called biocatalysts.

  • Enzymes are synthesized by living cells. The term ‘enzyme’ was coined by Kuhne (1878) for catalytically active substances previously called ferments.

  • Enzymes were actually found out by Buchner (1897) with the accidental discovery that fermentation of sugar is not only caused by living yeast cells but also yeast extract. The extract obviously possessed biocatalysts required for the process (Gk. en.- in, zyme- yeast).

  • Buchner (1903) also isolated the first enzyme. He was awarded Nobel Prize in the same year, 1903. There are numerous enzymes as every biochemical reaction is catalysed by a separate enzyme.

Biological Importance

(i) Thousands of chemical reactions are taking place in the body of a living organism.all of them are mediated by enzymes.

(ii) Enzymes are specialized catalysts that operate at biological temperatures.

(iii) enzyme mediated reactions do not require harsh treatment.

(iv) They are pH specific so that reactions requiring different pH operate in different parts of the body.

(v) As they operate under favourable conditions, enzymes force the organisms to live under favourable environment.

(vi) Enzymes are highly regulated. Their formation is controlled by separate genes. Activation and repression of genes allow certain enzymes to be functional or nonfunctional in cells.

Chemical Nature Of Enzymes

  • All enzymes are globular proteins (Sumner, 1926) with the exception of recently discovered RNa enzymes. Some enzymes may additionally contain a nonprotein group. Accordingly there are two types of enzymes, simple and conjugate.

  • Simple Enzyme It is an enzyme which is wholly made up of protein. Active site is formed by specific grouping of its own amino acids. Additional substance or group is absent, e.g., pepsin, trypsin, urease.

  • Conjugate Enzyme. It is an enzyme which is formed of tow parts- a protein part called apoenzyme (e.g., flavoprotein) and a nonprotein part named cofactor. The complete conjugateenzyme, consisting of an apoenzyme and a cofactor, is called holoenzyme. Active site is formed jointly b apoenzyme and cofactor.

Active Site or Active Spot

  • The whole of enzyme molecule is not active in catalyzing a chemical reaction. Only a small portion of it is active. It is called active site or active spot.

  • An enzyme may have one to several active sites.

  • An active site or spot 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. It fails to recognize other molecules.

Classification

In older times enzymes were classified into two broad categories.

(i) Hydrolyzing:

Catalyzing hydrolysis of large molecules into smaller ones, e.g., carbohydrates or amylases,proteases, lipases, esterases, phosphorylases, amidases. Diagestive enzymes are hydrolyzing in nature. They are often grouped intothree types- proteolytic, amylolytic and lipolytic.

(ii) Desmolysing:

Catalyzing reactions other than hyroysis, e.g., aldolases, dehydrogenases, oxidases, peroxidases, catalase, carboxylases, etc.

The modern system of enzyme classification was introduced by interational Union of Biochemistry (IUB) in 1961. It groups enzymes into the following six categories:

(a) Oxidoreductases

  • They take part in oxidation and reduction reactions or transfer of electrons.  

  • Oxidoreductases are of three types- oxidases, dehydrogenases and reductases, e.g.,

  • Cycytochrome oxidase (oxidizes cytochrome), succinate dehydrogenase, nitrate reductase.

(b) Transferases

They transfer a group from one molecule to another e,g., glutamate-pyruvate transaminase (transfers amino group from glutamate to pyruvate during synthesis of alanine). The chemical group transfer does not occur in free state.

(c) Hydrolases

They catalyse hydrolysisof bonds like ester, ether, peptide, glycosidic, C- C, C – halide, P – N, etc. which are formed by dehydration condensation. Hydrolases break up large molecules into smaller ones with the help of hydrogen and hydroxyl groups of water molecules. The phenomenon is called hydrolysis. Digestive enzymes belong to this group, e.g., amylase (hydrolysis of starch), sucrose, lactase.

(d) Lyases

The enzymes cause cleavage, removal of groups without hydrolysis, addition of groups to double bonds or removal of a group producing double, e.g., histidine decarboxylase (breaks histidine to glyceraldehydes phosphate).

(e) Isomerases

The enzymes cause rearrangement of molecular structure to effect isomeric changes. They  are of three types, isomerases (aldose to ketose group or vice- versa like glucose 6-phosphate to fructose 6-phosphate), epimerases (change in position of one constituent or carbon group like xylulose phosphate to ribulose phosphate) and mutases ( shifting the position of side group like glucose-6-phosphate to glucose-1- phosphate).

(f) Ligases (synthetases)

The enzymes catalyse bonding of two chemicals with the help of energy obtained from ATP resulting in formation of such bonds as C- O, C- S, C – N and P- O, e.g., pyruvate carboxylase. It combines pyruvic acid with CO2 to produce oxaloacetic acid.

Characteristics (Properties Of Enzymes)

  1. Protein nature. Enzymes are generally globular proteins. They may have additional inorganic or organic substances for their activity.

2) Molecular weight. Being proteinaceous, the enzymes are giant molecules with a molecular weight of 6000 (bacterial ferredoxin) to 4,600,000 (pyruvate dehydrogenase complex).

3) Colloidal nature. They are hydrophilic and form hydrosol in the free state.

4) Chemical Reaction. Enzymes do not start a chemical reaction but increase the rate of chemical reaction. They do not change the equilibrium but bring about equilibrium very soon.

5) Efficiency. The number of substrate molecules changed per minute by a molecule or enzyme is called turn over number (kcat). The higher the turn-over number, the more efficient an enzyme is.

6) Unchanged Form. Enzymes are in no way transformed or used up in the chemical reaction but come out unchanged at the end of reaction.

7) Reversibility. Theoretically, all enzyme controlled reactions are reversible. Reversibility is, however, dependent upon energy requirements, availability of reactants, concentration of end products and pH.

8) Enzyme specificity. Enzymes are highly specific in their action. For example, enzyme maltase acts on sugar maltose but not on lactose or sucrose. Different enzymes may act on the same substrate but give rise to different products. For example, raffinose gives rise to melibiose and fructose in the presence of enzyme an enzyme may act on different substrates, e.g., sucrose can act on both sucrose and raffinose producing different end products.

9) Heat Sensitivity. All enzymes are heat sensitive or thermolabile. Most enzymes operate optimally between 250 – 35oC.

10) Protein poisons. Being made of proteins, enzymes are inactivated or denatured by all those substances and forces which destroy energy radiations.

12) pH.  Each enzyme functions at a particular pH. e.g., pepsin (2pH), sucrose (4.5 pH), salivary amylase (6.8pH), trypsin (8.5pH). a change in pH makes the enzymes ineffective.

13) Enzyme- substrate Complex. The active sites of enzymes have a specific conformation for attracting and holding substrate. It usually possess a crevice or pocket where the substrate fits in a complementary fashion.

Factors Influencing Enzyme Activity

(1) Temperature. An enzyme is active within a narrow range of temperature. The temperature at generally corresponds to the body temperature of warm blooded animals,e.g., 37oC in human beings. Enzyme activity decreases above and below this temperature. Enzyme become inactive below minimum temperature and beyond maximum temperature.

(2) Optimum pH. Every enzyme has an optimum pH  when it is most effective. A rise or fall in pH reduces enzyme activity by changing the degree of ionization of its side chains. A change in pH may also start reverse reaction.

(3) Enzyme concentration. The rate of a biochemical reaction rises with the increase in enzyme concentration upto a point called limiting or saturation point.

(4) Product concentration. If the products are allowed to remain in the area of the Revers reaction can also start.

(5) Activators. They increase activity of enzymes (e.g., chloride for salivary amylase), function as cofactors (e.g., K+, Mn2+) and convert proenzymes to enzyme state.

(6) Protein poisons. Cyanides, azides, iodoacetate, and salts of heavy metals destroy tertiary structure of enzymes by either combining with cofactor or a group of apoenzyme (- SH group, -COOH).

(7) Radiation Energy. High energy radiations break hydrogen bonds, ionic bonds, and other weak linkages to destroy enzyme structure.

(8) Substrate Concentration. Increase in substrate concentration increases the rate of reaction. The enhanced rate is due to two factors:

(a) occupation of more and more active sites by the substrate molecules;

(b) higher number of collisions between subctrate molecules. The rise in velocity is quite high in the beginning but it decreases progressively with the increase in substrate concentration.

Michaelis Constant 

(Michaelis Menten Constant, Km). it is a mathematical derivation or constant which indicates the chemical reaction catalysed by an enzyme attains half its maximum velocity.

Activation Energy

Most of the chemical reactions do not start automatically because the reactant molecules have an energy barrier to become reactive. The energy barrier may be on account of :

(A) Mutual repulsion due to presence of electrons over their surfaces.

(B) Salvation or holding of reactants in solution  form by hydrogen bonds.

(C) Reaction sites of the reactive molecules being small, precise collisions do not occur. Therefore, an external supply of energy is needed for the start of the chemical reaction. It is called activation energy. Activation energy of the system and brings about forceful collisions between the reactants. The requirements of activation energy is quite high.

Mode Of Enzyme Action

There are two view points by which enzymes are supposed to bring about chemical reaction.

(1) Lock and key hypothesis. It was put forward by Emil Fischer in 1894. According to this hypothesis, both enzyme and substrate molecules have specific geometrical shapes. In the region of active sites the surface configuration of the enzyme is such as to allow the particular substrate molecules to be held over it. The active sites also contain special groups having – NH2, -COOH, -SH for establishing contact with the together causing the chemical change.

The products remain attached to the anzyme for some time so that an enzyme-product complex is also formed. However, the products are soon released and the freed enzyme is able to bind more substrate molecules.

Enzyme + Substrate ------- Enzyme – Substrate Complex

Enzyme – Substrate Complex ------ enzyme – products complex

Enzyme – Products Complex ------Enzyme + Products

(2) Induced-fit theory. It is modification of lock and key hypothesis which 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. The catalytic groups are normally at a distance. As soon as the substrate comes in contact with the buttressing group, the active site of the enzyme undergoes conformational changes so as to bring the catalytic group opposite the substrate bonds to be broken.

Differences between Lock and Key and Induced Fit Theories

S.No.

Lock And Key Theory

Induced Fit Theory

1.

Active site is a single entity.

Active site is made of two components.

2.

There is no separate catalytic group.

A separate catalytic group is visualized.

3.

Active site is static.

In contact with substratum, the butteressing group undergoes conformational change.

4.

Development of transition state is not considered.

It considers the development of

5.

It does not visualize the weakening of substrate bonds.

Catalytic group is believed to weaken the substrate bonds by nucleophilic and electrophilic attack.

6.

It does not explain the mechanism of non-activity in case of competitive inhibitor.

It gives a mechanism for nonaction over competitive inhibitor.

Catalysts And Enzymes

Catalysts are inorganic substances which increase the rate of chemical reactions without themselves undergoing any change and without modifying the equilibrium of the reactions. Enzymes are similar chemicals which are biological in origin and operate in the biochemical world.

Isoenzymes (Isozymes)

At one time it was believed that an organism has only a single enzyme for a given step of a metabolic reaction. It was later discovered that a substrate may be acted upon by a number of variants of an enzyme producing the same product. The multiple molecular forms of an enzyme occurring in the some organism and having a similar substrate activity are called isoenzymes or isozymes.

Inhibition Of Enzyme Action

Reduction or stoppage of enzyme activity due to presence of adverse conditions or chemicals is called enzyme inhibition. It is of several types. Inhibition can be classified into two

(a) Competitive and noncompetitive

(b) Reversible and irreversible

Competitive inhibition is caused by swamping of the active sites by a chemical which is similar in structure to the substrate but does not undergo chemical change. Competitive inhibition is usually reversible. Non – competitive inhibition is caused by alteration of conformation of the enzyme by a chemical that binds to a site other than the active site.   

Irreversible inhibition is of permanent nature as the enzyme conformation is harmed. Denaturation of enzyme is an example of irreversible inhibition. Reversible inhibition is that inhibition which can be overcome by withdrawal of the inhibitor because the effect of the latter is of temporary nature due to blocking of active site or binding to linkages required for maintenance of active site. Dilution and dialysis reduces or eliminates the effect of reversible inhibition.

(1) Protein Denaturation. Enzyme activity is dependent upon the maintenance of tertiary structure of the protein moiety.the latter is destroyed by several factors like heat, high energy radiatons and salts of heavy metals.

(2) Competitive Inhibition. It is the inhibition of enzyme activity by the presence of a chemical that competes with the substrate for binding to the active site of the enzyme. The inhibitor chemical is also called substrate analogue or competitive inhibitor. It resembles the substrate in structure and gets bound up to the active site of the enzyme without getting transformed by the latter.

(3) Non- competitive Inhibition. It is an irreversible inhibition of enzyme activity by the presence of a substance that has no structural similarity with the substrate. It is of two types, reversible and irreversible. The irreversible noncompetitive inhibitor destroys or combines irreversibility with a functional group of enzyme that is essential for its catalytic function.

(4) Allosteric Modulation or Feed Back Inhibition. It is a type of reversible inhibition found in allosteric enzymes. The inhibitor is noncompetitive and is usually a low molecular intermediate or product of a metabolic pathway having a chain of reactions involving a number of enzymes. It is, therefore, also called end product or feedback inhibition. The inhibitor is also called modulator.

Q.1- "Lysosomes" were discovered by [Kashmir MEE 1995; CPMT 1996; MP PMT 2003]

(a) Haekel  (b) De Duve  (c) De Vries  (d) Purkinje

Q.2- The "marker" enzyme of lysosome is [BHU 1991]

(a) Lysozyme (muramidase)  (b) Acid protease

(c) Acid phosphatase            (d) Beta-galactosidase

Q.3- Which of the following statements is incorrect with reference to lysosomes [JIPMER 2002]

(a) They are filled acid hydrolase and other enzymes

(b) They are monomorphic and uniform in structure and function

(c) They may be autophagic

(d) They can digest proteins, nuclei acids, lipids and polysaccharides

Q.4- Which of the following organ has single membrane [CBSE PMT 1999]

(a) Nucleus(b) Cell wall(c) Mitochondria(d) Spherosomes

Q.5- Lysosomes are rich in [CBSE 1996; CPMT 1998; MP PMT 2003]

(a) Polyribosome    (b) Lipoproteins

(c) DNA ligase       (d) Hydrolytic enzymes

Q.6- Lysosomes are generally found in [BHU 1990]

(a) Animal cells only      (b) Animal cell and in some plant cells

(c) Plant cells only         (d) Bacterial cells

Q.7- Which of the function is performed by lysosome [MP PMT 2002]

(a) Breakdown of cell substances   (b) Photosynthesis

(c) Breakdown of water                  (d) Synthesis of protein

Q.8- At which pH enzymes of Lysosomes are usually active [CPMT 2002]

(a) pH 5   (b) pH 7    (c) pH 8   (d) In any pH

Q.9- The organelles whose major function is storage of hydrolytic enzymes are

[DPMT 1986; CPMT 1987, 93; MP PAT 1995; MP PMT 1993, 2003]

(a) Centrioles     (b) Chromoplasts    (c) Lysosomes    (d) Chloroplasts

Q.10- A lysosome in which intracellular organells is getting digested is called

(a) Primary lysosome    (b) Secondary lysosome

(c) Autophagosome       (d) None of these

Q.1

Q.2

Q.3

Q.4

Q.5

b

c

b

d

d

Q.6

Q.7

Q.8

Q.9

Q.10

b

a

a

c

c

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