Enzymes: Quick Notes on Enzymes |Plant Physiology

Quick Notes on  Enzymes:- 1. Distribution of Enzymes 2. Characteristics of Enzyme 3. Factors Affecting 4. Classification 5. Aspects Concerning 6. Structure 7. Mechanism 8. Relationship between Enzyme Structure and Enzyme Activity 9. Enzyme-Catalysed Reactions 10. Assays 11. Isoenzymes 12. Multienzyme Systems 13. Allosteric Enzymes 14. Co-Factors.

Contents:

1. Distribution of Enzymes
2. Characteristics of Enzyme
3. Factors Affecting Enzyme Activity
4. Classification of Enzymes
5. Aspects Concerning Enzyme Activity
6. Structure of Enzymes
7. Mechanism of Enzymes
8. Relationship between Enzyme Structure and Enzyme Activity
9. Enzyme-Catalysed Reactions
10. Assays of Enzymes
11. Isoenzymes
12. Multienzyme Systems
13. Allosteric Enzymes
14. Co-Factors of Enzymes

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1. Distribution of Enzymes:

All enzymes are proteins and they are produced through translation inside the cell. However, they may function inside or outside the cells where they are synthesized. While the former are termed intracellular or endoenzymes the latter are called extracellular or exoenzymes.

Exoenzymes are widely distributed in bacteria and fungi and they secrete enzymes on their substrates in order to break down their complex organic compounds into diffusible and simple components. Because of the action of enzymes, simple chemical substances produced are easily absorbed and assimilated by these organisms.

Exoenzymes are also reported in insectivorous plants like Drosera, Pinguicula, Nepenthes, etc. In these plants there are special trichomes present on the leaves which secrete hydrolase(s) within the cytoplasm of the head and this is poured out through the pores in the cuticle.

These hydrolases e.g., acid phosphatases, esterases, proteolases, ribonuclease, etc., are used for the digestion of the insect organic materials. Similar examples are also found in pollen and pollen-stigma interaction. In fact the exine of pollen abounds in several hydrolases and other enzymes and they are used for the digestion of complex organic molecules on the stigma and also in recognition-rejection responses.

The scutellum of several cereals like barley, maize also secrete some enzymes especially hydrolases like amylase, etc. for absorbing metabolites from the endosperm which is rich in starch. The basic level of metabolic control exists at the enzyme level. The cell is compartmentalized into several organelles like plastids, mitochondria, lysosomes, etc.

Each of the compartments possesses its specific set of enzymes. In fact, enzyme system of an organelle comprises a group of specifically arranged enzymes contained in it and has the ability to carry on a sequence of reactions.

In general, compartmentalization has several advantages like separation of diverse metabolic reactions; separation of hydrolases in lysosomes or vacuoles and save the other cell constituents from undesirable hydrolysis; metabolism of toxic substances locally e.g. peroxisomes metabolise hydrogen peroxide. Mitochondria, chloroplasts and vacuoles possess large number of endoenzymes. In fact the former two organelles possess their own DNA complements.

Chloroplasts have set of enzymes which bring about photolysis of water, photophosph- orylation, CO₂ assimilation, synthesis and degradation of starch and sucrose, etc. On the other hand, mitochondria contain diverse enzyme types which are involved in citric acid cycle, electron transfer, phosphorylation, amino acid and protein synthesis, etc.

Enzymes of nucleus are chiefly involved in DNA replication, transcription and amino acid metabolism, etc..Glyoxysomes have the enzymes of β-oxidation of fatty acids to Acetyl-CoA. Peroxisomes have enzymes which transform glycolate into amino acids e.g. serine, glycine. Vaculoes have been shown to have several hydrolases involved in diverse metabolism.

Cytoplasm also possesses several enzymes which participate in amino acid and protein synthesis, glycolysis, pentose phosphate pathway, etc. Cytoplasmic matrix also has enzymes concerned with membrane biosynthesis, hydrolysis of several macromolecules. Some of the enzymes have been shown to be present in the wall.

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2. Characteristics of Enzyme:

According to Dixon and Webb (1964), an enzyme is defined as ‘a protein with catalytic properties due to its power of specific activation’. Enzymes act as catalysts since they increase the rate of a chemical reaction without an increase in temperature. Enzymes lower the level of the activation energy.

This is achieved by combining with the reactants to produce a transition state complex having lower energy of activation than the substrate. Once the product is formed the enzymes are released or regenerated and are used again. Compared with inorganic compounds, enzymes are more effective in reducing the activation energy.

It is suggested that most enzymic reactions, occur at 10⁸ – 10¹¹ times the rate of the corresponding non-enzymic reactions. A substrate is a substance on which the enzyme acts and activates it. In addition to the enzyme and substrate, several of the reactions require non-­protein structure or cofactor for the efficient catalysis of the reaction.

Cofactors are divisible into two groups: prosthetic groups which are tightly bound to the enzyme structure and these are easily separable from the enzyme i.e., coenzymes. During the enzymic reactions, coenzymes undergo some structural changes but in the later reactions they are regenerated.

There are a large number of compounds which are used to reduce the rate of an enzymic reaction and these are termed as inhibitors. Inhibitors usually should act at low concentrations and some of these are specific for certain reactions. However, strong acids or bases are excluded from such a list of inhibitors.

Specific activity of the enzymes is referred to as units of enzymes per milligram of protein. Molecular activity is expressed as units per micromole of enzyme at optimal substrate concentration.

It is also defined as the number of molecules of substrate transformed per minute per molecule of enzyme. According to the Internaitonal Commission on Enzymes, one unit of enzyme (U) is defined as that content which will catalyse the transformation of micromole of substrate per minute. It is important to state the temperature and preferrably reactions be done at 30°C. pH and substrate concentrations should be optimal.

The activity of enzymes is affected by large number of factors and some of the most important factors are pH, temperature, substrate concentration, cofactors and enzyme.

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3. Factors Affecting Enzyme Activity:

Substrate concentration:

The initial velocity of an enzyme catalysed reaction is directly proportional to the enzyme concentration. Thus, given more enzyme molecules at the reaction site, the rate of the reaction is accelerated. If the enzyme reaction at a given site is fixed, and the substrate content is enhanced, initially the reaction rate will rise enormously.

If the concentration is continued to increase, the rate of reaction will slow down, until no further change is noticed. Maximum velocity of the enzyme-catalysed reaction represents the velocity of the reaction obtained at high substrate concentration. The substrate concentration needed to yield half the maximum velocity is called Michaelis constant (K_m). The K_m value represents a valuable clue to the mode of action of an enzyme. It is also represented as follows: as Michaelis-Menton equation. K_m= K_s when the substrate concentration gives half the maximum velocity.

V = V_max/K_s + s

In this equation s is equal to K_s, V is equal to V_max. It may be stated that K_m is a characteristic of the enzyme which is independent of enzyme concentration but may be affected by pH and temperature.

If an enzyme catalyses the transformation of more than one substrate, it exhibits characteristic K_m for each substrate.

pH:

Nearly all enzymes are sensitive to changes in pH and have a pH optimum at which activity is maximum. pH affects enzymes activity in several ways. It causes change in protein structure, changes the ionization of the substrate, or changes the ionization of various groups in the enzyme molecule. pH may also alter the ionization of the enzyme-substrate complex. Some of these factors will affect K_m value while others will influence V_max of the reaction.

Temperature:

In most cases, rate of enzymic reaction increases with an increase in temperature, till a temperature is reached when the rate falls sharply.

This results from the interaction of the two. At high temperature enzyme protein is denatured.

Most enzymes are denatured rapidly much below 70°C though some enzymes can tolerate a temperature as high as 100°C.

Hydration:

Hydration level also affects enzyme activity. In general, cells which have high water content have enzymes which are fully hydrated. In mature seeds, spores, pollen grains, which have comparatively low hydration level, the enzyme activity is extremely feeble. In the highly dehydrated tissues the enzyme activity is negligible.

Redox Potential:

Redox potential of the medium also affects the enzyme activity. Thus, several of them have reduced sulphur (—SH) groups that must remain in their reduced form for the enzyme to function.

Enzyme Inhibition:

Large numbers of inhibitors have been used to decrease the rate of a biochemical reaction in a process. The nature and action of these inhibitors has provided invaluable information on the nature of the enzyme-substrate interactions and the mechanism of catalysis of enzymic reactions.

Some of the inhibitors have been successfully utilized to block a specific step in series of reactions in a process and yielded crucial evidence leading to the discovery of various metabolic pathways. Enzyme inhibitors are either reversible or irreversible. In the former, the enzyme activity is restored once the inhibitor is removed.

On the contrary irreversible inhibitors cannot be removed by dialysis and include alkylating agents. Most of the inhibitors used are reversible and are classified into two main types: competitive and non-competitive depending upon whether they compete with the substrate for the enzyme or whether they combine with the enzyme in some way to reduce its activity. There are also uncompetitive and mixed inhibitors.

The most well quoted example of competitive inhibitor is malonate which causes competitive inhibition by inhibiting succinate dehydrogenase.

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4. Classification of Enzymes:

International Commission on Enzymes has adopted a standard classification and nomenclature of the enzymes. According to this scheme, the enzymes are grouped into six major classes taking into consideration the chemical reaction that they catalyse. Each enzyme is named after adding ase to its substrate and this describes the type of reaction carried out by that enzyme.

The major classes are given number from 1 to 6 and three other names denoting the various sub-classes. Some enzymes are also given short or trivial names. Thus, acid phosphatase is a trivial name for orthophosphoric monoester phosphohydrolase and is accorded the number 3.1.3.2.

First number indicates its major class which is hydrolases, the second the subclass of the hydrolases which acts on ester bonds, the third number is sub-subclass that hydrolyses phosphoric monoester. The fourth figure (2 in our example) is the serial number of the enzyme.

Based on the classification proposed by Dixon and Webb, following six major classes are recognized:

1. Oxidoreductases:

The enzymes included in this class catalyse oxidation-reduction reactions and are thus associated with respiration in the cell.

Dehydrogenases, oxidases are included in this class. Dehydrogenases include succinate, malate, glutamate, isocitrate dehydrogenases.

They cause oxidation in conjunction with co-enzymes like NAD and NADP which act as hydrogen acceptors. Peroxidase is an oxidase which uses H₂O₂ as an oxidant.

2. Transferases:

These enzymes cause the transfer of various groups; for instance, one-carbon group, and phosphate group from substrate to acceptor molecule.

3. Hydrolases:

It includes several of the enzymes which catalyse a wide range of hydrolytic reactions involving the introduction of a water molecule.

This class includes enzymes that hydrolyse ester linkages, glycosidic bonds and peptide bonds.

AB+H₂ O → AH + BOH

4. Lyases:

Enzymes included in this class catalyse the addition of groups to double bonds or vice versa. This class includes decarboxylases, aldolases etc.

5. Isomerases:

Enzymes in this class bring about isomerization and include epimerases and other enzymes.

6. Ligases:

The enzymes of this class catalyse the linking of two molecules coupled with breakdown of ATP or other triphosphates. Thiokinase is example of the enzyme of this class.

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5. Aspects Concerning Enzyme Activity:

Activation:

When a substance is added to an enzyme solution, catalytic activity is enhanced and the process is called enzyme activation.

The substance which causes such an increase is termed activator. The enzyme activation can take place in several ways including (i) conversion of zymogen to active enzyme; maintenance of groups (—SH) in an enzyme; stimulation of enzyme by cofactor and allosteric effect or stimulation by several regulatory or allosteric enzymes.

Specificity:

Enzymes are highly substrate specific i.e. their action is restricted to a specific singular substrate or a few closely related substrates. This characteristic maintains orderly metabolism within a cell. However, the degree of specificity exhibited by an enzyme varies considerably.

The specificity may be absolute (i.e. acting only on one substrate), they may show group specificity (i.e., act on several substrates which, have a specific structure in common). Enzymes also show marked stereo-specificity i.e. they attack only one of the optically active isomers. Some enzymes can distinguish between chemically identical groups in symmetrical molecules.

Information pertaining to enzymes specificity is vital for understanding the nature of the enzyme- substrate complex. Higher specificity indicates greater chances of joining of the binding sites of the two structures.

It is also possible to replace specific groups on the substrate molecules by a series of other groups and thus determine the nature of the functional groups. The general deduction is that enzyme specificity is determined by two structural features of the substrate molecules: one, the distinct chemical bond attacked by the enzyme and the other functional groups in the substrate.

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6. Structure of Enzyme:

Basically all enzymes are protein molecules though sometimes additional prosthetic group may also be attached which acts as an efficient catalyst.

The molecular weight varies from 10,000 to over one million and, therefore, contains several thousand amino acids.

Comparatively the substrate molecules are very small and, therefore, a very small portion of the polypeptide chain is in contact with the substrate in the enzyme-substrate complex.

Such observations have given a concept of active site of the enzyme i.e., it is the part of the enzyme molecules at which the process of activation and reaction of the substrate occurs and this site is so made to allow the specific substrate molecule.

Some workers have proposed that enzymes in general contain three major components of structure and these are— functionally active centre, the residue which supports and stabilizes these centres, and the part of the amino acids which lend structural framework on which the functional portions are arranged.

Some enzymes are unduly large and some bits of them can be removed without causing any alteration in their specificity or activity.

For proper understanding of the enzyme reaction, it is essential to identify the amino acids associated with the active centre.

The functional amino acids are identified by several methods which include labelling of the active centre (i.e., diopropylflurophosphate to label active centre in proteases and esterases), inactivation of enzyme by the modification of certain residues etc.

Lack of specificity can also be overcome by affinity labelling. In addition kinetic studies can also provide vital information pertinent to the nature of the active centre. Some workers have also used X- ray diffraction methods to elucidate the mechanism of enzyme action.

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7. Mechanism of Enzyme Action:

Enzyme action involves the formation of the complex between enzyme and its substrate. In some enzymes there is a direct chemical evidence for this combination. Based on the precision of enzyme- substrate complexing an analogy with lock and key was proposed.

In this hypothesis it is assumed that substrate like a key must have a specific shape to fit into the specific hole on the enzyme surface. If this concept is accepted several corollaries emerge thereby. Firstly, the interaction with the substrate could be proposed as involving the three-dimensional structure of the molecule and also catalytically significant groups.

Good amount of experimental evidence support this contention. Accordingly once the tertiary structure of enzyme is disrupted they no more retain their catalytic activity. Several authors have also suggested that highly specific enzymes must have three points of attachment to the substrate.

This also helps them to discriminate between substrates which are stereoisomers. Secondly, the concept of close spatial fit between enzyme and substrate furnished a structural basis for the action of competitive inhibitors.

This inhibitor possibly occupied a part of the substrate hole on the enzyme. Thus, the inhibitor competed with the substrate molecules for association with the enzyme. End products if remain attached to substrate also act as competitive inhibitors. In situations where the end product level in the medium is low, it dissociates from the enzyme.

Based on the fact that some of the enzymes seem to undergo small structural alterations when complexed to the substrate calls for the new thinking on enzyme-substrate combination. Thus Koshland (1959) proposed induced-fit hypothesis. In his viewpoint enzyme assumingly undergoes some structural distortion following its association with substrate.

Recently Phillips has studied enzyme lysozyme and obtained amino acid sequence based on the orientation of the polypeptide chain lysozyme crystals. Phillips also obtained some definite information on the nature of physical association between enzyme and substrate. Using competitive inhibitors this worker identified the catalytic centre.

The crystallized enzyme was subjected to X-ray studies and he observed that lysozme’s substrate binding site was like a cleft in the three-dimensional structure of the enzyme. According to Phillips, the enzyme is having the region or binding the substrate which could accommodate six ammo-sugar units of the polysaccharide substrate.

He also reported that binding involved distortion of the enzyme as well as substrate structure. Indeed such a step helped in the splitting of the substrate. Subsequently another type of enzyme-substrate combination was brought out with regard to carboxypeptidase. Lipcomb proposed the detailed combination based on his X-ray crystallographic studies on this enzyme.

Here again binding of the substrate caused considerable modifications in the enzyme shape. Substrate also altered the position of many of the amino acids residues at the active centre. Based on his studies it seems clear that an enzyme-substrate could only be formed when the substrate induces a change in the enzyme configuration.

Such type of mechanism is termed as embracing mechanism. It may be added that precise mechanism of action of carboxypeptidase is not vivid though it seems to involve the zinc atom, glutamic acid 270, tyrosine 248, etc. Recently some X-ray diffraction studies have been carried out in chymotrypsin and the available information has provided a third variation of the enzyme-substrate combination.

Functionally the enzyme is similar to carboxypeptidase but possesses different specificity. Blow and Steitz (1970) have proposed a model of this enzyme. They have obtained some information on what gives the enzyme its specificity.

Here Tryptophan sidechain fits into a Hydrophosic without any distortion of the enzyme. Consequently the nitrogen of the peptide bond which needs to be split comes close to the portion which forms the hydrogen bond between Histidine 57 and Serine 195. This arrangement of the enzyme is vital for its hydrolytic activity. In fact, the mechanism is a two-step process.

Firstly, there is acylation of Serine 195 to release the amino terminal end of one of the newly formed peptide fragments and then deacylation to release other peptide. In the whole association of enzyme substrate there is no alteration of the enzyme configuration.

The catalytic property seems due to a specific position in relation to the serine and Histidine at the active centre of the enzyme. This enzyme evidently confirms to the lock-and-key view point of enzyme-substrate combination (Fig. 5-1).

From the above discussion, it becomes clear that X-ray crystallographic studies have provided three viewpoints on the mechanism of enzyme-substrate combination.

These are:

(i) Substrate may fit in the hole of a particular shape on the enzyme e.g. chymotrypsin;

(ii) The enzymes can form a productive complex with a substrate which causes sufficient distortion to make the enzyme act as a catalyst e.g. lysozyme;

(iii) An appropriate substrate may induce an active configuration in the enzyme by causing a change in the configuration of the enzyme e.g. carboxypeptidase.

From the above discussion it is apparent that a specific orientation of the substrate on the enzyme molecule is very significant. Thus the part of the substrate which participates in the reaction comes in close association with the reactive group on the enzyme. It is the characteristic feature of the group which ultimately determines the catalytic effect.

In some instances it is the amino acid side chain behaviour, which may be acidic or basic, which makes the proteins effective catalysts. Thus, the reactive groups on the enzyme may stimulate the action of non-protein catalysts which are electrophilic or nucleophilic. An electrophilic group is an acidic one and can act as an electron donor e.g. Mn²⁺, Fe³⁺.

On the other hand, the nucleophilic group is an electron donor and several of the protein side chains act like this. A nucleophilic group can readily react with an electrophilic group. This type of reaction is called nucleophilic displacement. In case of a carboxypeptidase and lysozyme a catalytic effect based on the nucleophilic feature of one or more of the side chains at the active centre has been proposed.

It seems that an ionized group of glutamic acid or aspartic acid serves as the nucleophilic group and donates electrons to affect a displacement reaction with the substrate. In lysozyme these are aspartic acid residue 52 and glutamic 35 while for carboxypeptidase it is glutamic acid 270 which acts as the nucleophilic group and tyrosine 248 as a proton donor.

However, for the two enzymes the nucleophilic effect is accompanied by an electrostatic effect which causes a steric straining of the enzyme and the substrate due to their combination. In summary, we may mention that all the three enzymes produce the same reaction or the general effect i.e. they cause hydrolytic action. Obviously different enzymes have different mechanisms of operation.

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8. Relationship between Enzyme Structure and Enzyme Activity:

Recent studies on enzymes have indicated remarkable complexity of relationship between enzyme structure and function since the enzyme comprises several protein subunits. In general a few common points may be noted. Any enzyme may have several subunits and each subunit may have its own catalytic centre.

The association of the substrate-enzyme is dependent upon three dimensional shape of the catalytic centre as its position. In fact, there are two forms of a molecule e.g. one where catalytic subunit can easily bind to the substrate and the other where very little or no binding can occur. Also binding of the substrate to each of the catalytic sites stimulates the conversion of less active site of more active one.

It may also be stated that the binding of the first molecule increases the tendency of the enzyme to bind the subsequent one. This interrelationship between substrate binding sites is called cooperative interaction.

Some enzymes have two or more receptor sites and these are non-overlapping and stereo-specifically different. The sites which are other than the active sites are called allosteric sites. These sites can attract different substances and produce a reversible change in the structure of the enzyme.

This is termed as allosteric transition. Some of them reduce the active functioning of the active spots or sites. Substances which activate the sites are known as allosteric activators. Allsosteric enzymes are made up of similar types of subunits and are oligomers.

Enzyme-Catalysed Reactions:

A comparison can be made between a chemical reaction and enzyme- catalysed reactions. In a chemical reaction:

Here the rate of velocity, v of the forward or reverse direction is proportional to the reactants concentration i.e. v₁ = k₁ (A) (B) or v₂ = k₂ (C) (D), k₁ and k₂ are individual rate constants. At equilibrium, v₁ = v₂ and hence k₁ = k₂.

k₁/k₂= (C)(D)/(A)(B) = k_eq; k_eq = e, equilibrium constant.

The presence of an enzyme accelerates the accomplishment of the equilibrium state. For the completion of reaction, A and B must collide in the right way to allow product formation.

Closer the reactants rapid is the reaction. Enzymes promote the reaction by absorbing the reactants on to a polypeptide chain close to each other resulting in an effective concentration of the reactants.

A and B must collide with sufficient kinetic energy for the reaction to take place and this energy is called energy of motion.

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9. Assays of Enzyme Activity:

In an enzyme its catalytic activity is assayed by incubating a substrate of known amount with a suitable enzyme (E), at a pertinent pH for an appropriate time.

Then the end product (EP) is formed or substrate (S) disappears. These measurements are done through spectrophotometer.

The activities of enzymes which use coenzymes like NAD or NADP in oxidized or reduced state, assaying is done at 340 nm.

The activity of an enzyme is expressed in:

i. Katals. One katal is the amount of activity that converts 1 mol of substrate per second. Activity is expressed as µ kat or nanokatals (nkat).

ii. International units (IU). One IU is defined as the amount of enzyme which catalyses the transformation of 1 µmol of substrate per min under standard conditions.

iii. Specific activity, kat kg^-1 of protein or IU mg^-1 of protein.

iv. Molar activity, kat mol^-1 of enzyme.

Michaelis-Menton Equation:

This equation describes the interrelationship between the rate of an enzyme reaction and the concentration of substrate and enzyme.

Three assumptions are made:

i. The enzyme-substrate is in equilibrium with free enzyme and substrate in solution i.e. E + S ⇋ES

ii. The formation of this complex is essential for product formation i.e. ES → P + E

iii. The enzyme-substrate concentration is constant. With a view to defining a genetal expression for v₀, the initial velocity of the reaction, Michelis-Menton derivation is done as under:

The initial rate of product formation, v₀ = k₂ (ES). It is difficult to measure k₂ and (ES), therefore an alternate expression for v₀ in measurable variables (E) and (S) must be reasoned. Thus the further derivation is for ES formation:

d(ES)/dt = K₁ (E) (S)

It is advisible to consider the concentration of free and combined forms of the enzyme.

d(ES)/dt = K₁ (E_t) – (ES) (S)

E_t = total enzyme

when the breakdown of ES in forward and reverse directions is considered.

-d(ES)/dt= K_-1 (ES) – K₂ (ES)

In a steady state (ES) is constant, hence rate of ES formation = rate of ES breakdown:

K₁(CE_t) – (ES) (S) = K_-1 (ES) + K₂ (ES)

By grouping constants:

((E_t)-(ES))(S)/(ES) = K_-1 + K₂/K₁

The rate constant grouping is shown as K_m, the Michaelis constant.

v₀ = V(S)/K_m+(S)

This is Michaelis-Menton equation.

This equation relates the components of an enzymic reaction (S) and (E) to velocity, initial and maximum, through a rate constant (K_m). V = maximum velocity of the reaction and V is dependent upon (E).

When reaction rate is 50% of maximum velocity, then

v₀ = V/2

v₀ = V(S)/K_m+ (S)⇒ V/2 = V(S)/K_m+ (S)

Division by V gives

1/2 = (S)/K_m+(S)

and cross multiplication gives

K_m + (S) = 2(S), .^.. K_m = (S)

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10. Isoenzymes:

The term indicates multiple molecular forms of an enzyme sharing a catalytic activity, derived from a tissue of a single organism.

The term isoenzymes are a synonym often used by the biochemists. In general, isoenzymes having common catalytic activity may be widely differing enzymes synthesized under control of different genes, active in different tissues and differing in molecular properties. Isozymic variations may also arise from allelic segregations at a single locus representing more subtle changes in the enzyme molecule.

Isoenzyme analyses have been made in several enzymes in many plant species e.g. peroxidase, catalase, amylase, esterase, acid phosphatase, dehydrogenases etc. Molecular forms of an enzyme can be separated by sedimentation, electrophoresis, chromatography, gel filtration and even serological methods.

Of these gels electrophoresis is most widely used and is easy to operate. Electrophoresis is basically a process of forced diffusion within an electrical field. In this the protein molecules of the given sample are moved through a gel, paper or cellulose using electrical gradient.

Different proteins assume different charges, at different pH and depending upon their molecular weight they move on the gel. This results in separation of different bonds which can be stained and differentiated. Several staining procedures are available. Thus it is possible to obtain visible fingerprints or zymograms of an enzyme’s isoenzymes.

The compositions of media, buffers through which molecules must pass, contribute to the efficiency of isozyme patterns. In general, acrylamide and starch gels provide non-ionic framework for electrophoretic studies. Of the two, acrylamide gels are more useful and can be prepared at different concentrations and can withstand wide range of pH.

They are also well known for optical clarity. Through experience it is possible to adjust their concentrations and also depending upon the tissues which are to be studied. Electrophoretic studies have demonstrated that isozyme patterns and intensities are tissue and plant specific and also different developmental stages have specific isozyme patterns.

However, several of the factors like mineral nutrition, cold temperature hardening, disease infection and injury affect the intensity of isozymes. Thus in any isozyme investigation it is always advisable to compare tissues which have similar developmental stage.

Moreover, sufficient amount of sample must be obtained to procure clear bands. Similarly sample size, gel composition, pH, electrolyte play an important role in getting clear separation of isozyme bands.

Isoenzyme studies can be fruitfully employed for identifying differences between taxa. Mendelian segregations of isozymes, work out probable evolutionary pathways in species complex or related genera and families, and associate isozyme patterns with external stresses which affect plant growth.

Similar correlations of isozyme patterns with basic metabolic and physiological phenomena may also be obtained.

In recent years isozyme patterns have been associated with tissue differentiation and physiology, the mechanism of disease resistance and certain other aspects of disease physiology of fungal pathogens.

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11. Multienzyme Systems:

These enzymes are sequential chains of catalysts involved in reactions where product of one reaction becomes substrate for next in the sequential chain.

Groups of individual enzymes which are physically correlated to function together are called enzyme complexes.

Here individual enzyme is inactive but whole set of enzyme function together convert precursor into a specific molecule.

One good example is synthesis of fatty acid from two-carbon precursors. Multi-enzyme systems associated with membranes are highly complex. Conventional biochemical techniques limit their detailed analyses.

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13. Allosteric Enzymes:

Some enzymes do not exhibit a hyperbolic curve when their initial velocity is plotted as function of substrate concentration.

Instead they show a sigmoidal curve. The rate of reaction catalysed by these enzymes at a given (S) is enhanced by the addition of a specific activator; and decreased by the addition of a specific inhibitor., and accordingly the curve tends to become hyperbola and sigmoidal under the two situations, respectively.

The enzymes which show this behaviour are called allosteric enzymes. Most allosteric enzymes possess sites other than catalytic site. On these additional sites that allosteric effectors are bound and influence catalytic events. Most allosteric enzymes possess several subunits e.g. pyruvate carboxylase.

Tracers are also effectively used in the study of plant enzymes. In this technique labelled compounds are employed in routine enzyme assays and also in studies of the kinetics and regulatory properties of enzymes.

The advantage of radiochemical assays includes their high sensitivity and specificity. These allow assaying of crude plant extracts even in the presence of inhibitors or contaminating enzymes that would interfere with a less sensitive assay type.

However, there are several potential disadvantages of radiochemical techniques. First, lot of time is involved and also it involves lot of efforts to separate labelled products from the labelled substrates especially if the solubility properties of the net electrical charges of these compounds are not different to allow rapid separation by solvent extraction or ion exchange chromatography.

Loewus and Labarca (1973) and Dickinson (1975, 1979) have analysed enzymes and metabolic pathways by which starch and cell wall polysaccharides are formed. These workers were especially keen to identify the enzymatic regulatory mechanism involved in germinating Lilium pollen.

Maize endosperm was also used as a system. Here the substrates were labelled with¹⁴C and the amount of labelled product was determined by scintillation counting, generally after rapid separation of product from unreacted substrate on ion- exchange paper.

This technique is advantageous since a small sample of the plant material can be used. Also only part of the plant tissue or plant can be used and the rest could be raised to maturity. Based on this methodology, Dickinson (1975) studied enzymes concerned with the formation of plant cell wall polysaccharides.

These were grouped under three categories:

(i) Reactions from simple sugars to glucan and galactan (hexokinase; phosphohexoseisomerase; phosphoglucomutase; UDP-glucose pyrophosphorylase; UDP-glucose epimerase; β- glucan synthase and galactan synthase).

(ii) Reaction from UDP-glucose to polymers containing uronic acid or pentoses (UDP-glucose dehydrogenase; UDP-glucuronateepimerase; UDP-glucuronate decarboxylase; UDP- arabinose epimerase; polysaccharide synthase, etc.).

(iii) Reaction from glucose-6-phosphate to UDP-glucuronate via myoinosital (myoinositol- phosphate synthase; myoinositol phosphate phosphatase; myoinositoloxygenase; glucuronokinase and UDP-glucuronicpyrophosphorylase).

Methods employed for the detection of enzymes in different plant tissues are by indirect experimental evidence i.e. in which a tissue metabolizes a substrate or forms a specific non-metabolic product. These assays are usually done by reacting the system (e.g. pollen) or a buffered extract with a specific substrate in the presence of redox (tetrazolium salts) or other dyes.

Another technique involves enzyme detection by placing the tissue on a known substrate and determining the change in respiratory gas exchange. Sometimes radioactive sugars are supplied to the tissues and labelled products detected by isolation and chromatography. By analysing the whole range of metabolic intermediates the enzymes involved are deduced.

The direct methodology is to extract and purify the different enzymes or through substrate metabolism experiments done on the homogenates. Based on enzyme assay, the metabolic conversions are recognized. The detection of enzyme activities suggests that assay of an isolated enzyme is subjected to considerable error and may be influenced by available combining sites by activating compounds present in the extract.

The sources of variation may be isoenzymes, extraction procedures, destruction and release of enzymes, diverse types of inhibitors in the system, etc. In general, histoenzymological procedures usually facilitate intracellular enzyme sites while direct enzyme assay of the particulate cell fractions isolated through differential centrifugation is also useful. However, both methods (quantitative and qualitative) have limitations in certain ways.

The ultimate role of different enzymes is to facilitate different processes. At a molecular level, enzymes are the key mediators for translating information from the nucleus and turn it into growth and metabolism. A detailed information on the locale and amount of enzymes would help in recognizing the regulatory mechanisms which influence morphogenesis of a particular system.

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14. Co-Factors of Enzyme:

Several of the enzymes additionally require a non-protein substance for the catalytic reaction to take place.

These substances are called cofactors and are classified into two major groups:

1. Activators:

These are simple inorganic substances e.g. magnesium and potassium ions which affect the enzyme conformation producing catalytically a more active state.

In the following table we give ion requirements of some enzymes. Some of the enzymes contain the metal e.g. alcohol dehydrogenase Zn⁺⁺; peroxidase, cytochromes (Fe⁺⁺); cytochrome oxidase (Cu⁺⁺).

2. Coenzymes:

These are complex organic compounds which are highly specific for a specific reaction; they are carrier of specific chemical groups.

During the course of a reaction; they may be structurally changed or regenerated in subsequent reaction.

Some of the coenzymes are tightly bound to the enzyme molecule and called prosthetic groups. Most coenzymes are loosely bound.

Some of the coenzymes like NAD and ATP move from one enzyme to another to fulfill their catalytic functions.

Adenosine Triphosphate:

It is abbreviated as ATP and is made up of a purine base called adenine and ribose sugar. The three phosphate groups are present and are attached in a chain to ribose part of adenosine. Of the three the last two phosphate radicals are energy rich. Each radical can provide nearly 11,500 cal per mole.

These are connected by energy rich bonds and the last radical can be easily dissociated or built up since it has a high transfer potential. ATP is best suited to trap and store chemical energy. The energy is also released with the same rapidity and is utilised in driving several reactions. It is used as an energy currency by the living systems.

Also ATP is an important phosphorylating agent which provides an active phosphate group to a specific metabolite. This phosphorylated metabolite then becomes highly reactive and can enter reactions where it could not participate initially.

Under normal circumstances ATP is produced from ADP and inorganic phosphate (Pi) and the reaction is endergonic. This process is called phosphorylation. At least three types of phosphorylations are known. These are substrate level phosphorylation, oxidative phosphorylation and photophosphorylation.

In the substrate level phosphorylation a phosphate group having high transfer potential is transferred from a phosphorylated compound to ADP. This type of phosphorylation takes place during respiration. In oxidative phosphorylation reduced coenzymes (NADH + H⁺) (FAD H₂) or succinic acid are oxidized.

The electrons obtained from these compounds pass over a series of electron carriers called respiratory chain. It is chiefly made up of cytochromes. Electron transport and phosphorylation occur at the same time. Photophosphorylation is similar to oxidative phosphorylation.

Uridine Nucleotides:

These coenzymes have a central role in carbohydrate metabolism of plant cells. UDP-sugar coenzymes play several important roles in carbohydrate metabolism and these are transformation of the sugar molecules themselves through the action of epimerase. They function as glycosyl group donors in the synthesis of different carbohydrates.

Pyridine Nucleotides:

Two pyridine nucleotide coenzymes act as hydrogen carriers in several of the oxidation-reduction reactions catalysed by the dehydrogenases. One of these is nicotinamide adenine dinucleotide {NAD) and comprises one molecule of nicotinamide, two molecules of ribose, one of adenine, and two of phosphate.

In the past this coenzyme was called diphosphopyridinenuceleotide (DPN) or coenzyme I. The second coenzyme is nicotinamide adenine dinucleotide phosphate (NADP). This coenzyme carried additional phosphate and was also called triphosphophopyridine (TPN) or coenzyme-II. Cells showing oxidation-reduction reactions invariably have these coenzymes. Generally, the enzymes are specific for either NAD or NADP.

Flavin Coenzymes:

These coenzymes are involved in oxidation-reduction reactions, and are tightly bound to the enzymes. They are regarded as prosthetic groups. There are two flavincoenzymes:flavin mono nucelotide (FMN) and flavin adenine dinucleotide (FAD). Nearly 40 flavoprotein enzymes have been described to date and involve variable substrates.

Haem Coenzymes:

Cytochromes, catalse, peroxidase and chlorophyll pigments contain haem groups as an essential functional part of the molecule.

Haem group comprises a porphyrin ring complexed with various metal ions. These coenzymes are concerned with the reactions including transport of oxygen, oxidation of substrates by hydrogen peroxide, and transport of electron to molecular oxygen in biological oxidation reactions.

Coenzyme A:

It is also called 3-phospho-ADP-pentonyl- β-alanyI-cysteamine and is ubiquitous to living cells, involved in reactions concerning the transfer of acyl groups. It is involved in oxidation of pyruvate by the TCA cycle.

Biotin:

It is found both in plant and animal tissues and is referred to as vitamin H. It concerns reaction5 involved in transfer or incorporation of carbon dioxide.

Thiamine Pyrophosphate:

This is also vitamin-associated coenzyme. Vitamin or thiamine functions as a coenzyme in the form of its pyrophosphate ester. The coenzyme is associated with synthesis or breakdown of carbon-carbon bonds adjacent to a keto group.

Tetrahydrofolic Acid:

This coenzyme participates in the reactions concerning transfer of one-carbon fragments.