Enzymes and Digestion in Plant (With Diagram)

The below mentioned article provides an overview on the enzymes and digestion process in plant.

Digestion in Plant:

In order to understand fully the true functions of raw materials which are taken in by the growing plant, it is necessary to know the mechanism by which the raw materials are elaborated into complex organic substances. Carbohydrates are synthesised in photo­synthesis.

From the carbohydrates produced, the plant is capable of manufacturing a host of other organic compounds of diverse nature known to be present in the plant, directly or indirectly.

There, in the tissues of plant, synthetic processes are continually being carried on and at the same time sugar and other substances are continuously being broken down into simpler constituents.

The chemical reactions resulting in the synthesis and degradation of complex organic substances are known as plant metabolism. The synthesis of complex organic substances from raw materials is usually called anabolism, the breakdown of those substances to simpler, organic or inorganic substances, catabolism.

The anabolic reactions are generally energy absorbing, i.e., endothermal or endergonic and catabolic, energy releasing, i.e., exothermic or exergonic. (If only the heat of the reaction is taken into account the terms endothermal and exothermal are used—in some cases heat of reaction and energy change are almost equal.)

From the point of view of the second law of thermodynamics, exergonic reactions are spontaneous. The endergonic or endothermal reactions, on the other hand, cannot take place unless free energy is supplied from some external source.

It must be clear, however, that many chemical reactions are taking place in the plant at any one time. Therefore, it is natural to assume that most of the endergonic or energy-absorbing processes are taking place at the expense of released free energy by the exergonic processes.

In general then, the anabolism of plants in which energy is absorbed is possible only if catabolism is simultaneously taking place. It is, however, true that free energy needed to drive some endothermal reaction may be obtained from sources other than exothermal reactions.

The most fundamental endothermal biochemical reaction taking place in plants, i.e., photosynthesis is undoubtedly driven by the free energy obtained from sunlight. Much of the food produced or accumulated in various parts of plant body is stored in the form of large organic complex molecules.

The conversion of these complex substances, usually insoluble, into simpler soluble forms is known as digestion. Some of the commonest of these temporary storage pro­ducts are starch, fats and proteins.

The most familiar digestive reaction in plants is the conversion of starch into sugar which is a hydrolytic decomposition or hydrolysis, i.e., breaking down into simpler forms by combination with water.

The significance of digestion lies in the fact that the simple products of digestion are usually more soluble, more diffusible, are more readily transported from cell to cell and are also more directly available for utilisation by cells.

Thus, starch which is not directly available for utilisa­tion and assimilation by the living cell is changed in all these respects by the process of digestion.

Many other storage compounds such as a host of complex carbohydrates, proteins, fats and other organic compounds, temporarily stored in more specialised storage organs of seeds, roots leaves, stems (underground stems), etc., become more directly available to growing cell through digestion.

This digestion is brought about by specific substances or organic catalysts that are commonly called anzymes. Again, gradual and orderly release of energy by the exergonic reactions, such as respiration at the right time and in the right place enables the plant to perform all its energy-consuming processes, such as active absorption of solutes, proto­plasmic streaming, cell growth, etc.

And none of these energy-consuming processes would have been possible in the absence’ of enzymes. Because the enzymes are highly active, they easily reveal their presence. It can be easily demonstrated that a substance which will not break down of itself, may readily do so when mixed with a plant juice. This indicates that the plant juice contains an enzyme that can catalyse the breakdown of the particular substance.

Thus a complex substance may be broken down step by step to simpler and simpler substances and at each step a packet or certain amount of energy is released which may easily be used to drive an endergonic or energy absorbing reaction.

The term enzyme was given to these substances by Kuhne in 1878. In 1860, Pasteur was the first to explain the phenomena of alcoholic and lactic acid fermentations and to think that they must be due to the vital activities of living cells.

It was later found that it was not the living cells themselves which brought about the decomposition of sugar but actually the breakdown was accomplished by certain chemical substances secreted by the living cells.

This was later fully confirmed when it was shown that a chemical sub­stance extracted from yeast converted large quantities of sugars into alcohol in the total absence of living cells.

Because the enzymes are very similar, although more complex in their activity to the inorganic catalysts, it is assumed that they are required only in extremely small quanti­ties.

It has been estimated that a single molecule of the enzyme catalase can effect the decomposition of approximately 5,000,000 molecules of hydrogen peroxide per minute, even at O°C.

Invertase, the enzyme which catalyses sucrose, can cause the breakdown of sucrose, 1,000,000 times that of its own weight without exhibiting appreciable diminu­tion in its activity.

Enzymes which operate within the cells in which they are produced are sometimes called endoenzymes. In rare cases the enzymes are excreted outside the cell and accom­plish their effects in external medium.

These are called exoenzymes. Metabolism of many bacteria and fungi grown in culture medium containing starch is due to the excretion of enzymes into the medium shown by gradual digestion of the medium.

Properties of Enzymes:

The number of known enzymes runs into hundreds and is increasingly daily. About 100 have been sufficiently purified and concentrated to obtain in crystalline state. All such enzymes have proved to be proteins or protein complexes of high molecular weights, ranging from 12,000 (Ribonuclease) to over 4,000,000 (urease).

Solubility:

Enzymes are soluble in water, dilute glycerin, alcohol, saline and buffer solutions.

Specificity:

Each enzyme may be pictured as having a definite shape into which only certain other molecules will fit like pieces in a jigsaw puzzle. The enzyme is able to piece together or to split up only those molecules which do fit it precisely.

Each indivi­dual enzyme is thus restricted in its catalytic activity to one particular reaction or one group of related chemical reactions. Proteolytic enzymes will thus act only on proteins: zymase will only ferment certain monosaccharides, not all.

Examples of extreme specifi­city are afforded by the enzyme, invertase which will act only on sucrose and not even on any other disaccharide; the enzyme urease only acts on urea, hydrolysing it into CO₃ and NH₃.

In some cases an enzyme may show specificity for a particular chemical linkage, as for example, the enzyme lipase which catalyses the cleavage of ester linkages in fat, without any particular regard for the nature of fatty acid involved in a fat.

Sensitivity:

Enzymes are very unstable in nature. Due to their protein nature, enzymes are heat labile. They are readily inactivated or destroyed by heat (60-100°C.) or by long exposure to light (particularly to ultra-violet or lights of still shorter wave­lengths).

Enzymes are inactive at temperatures near freezing point but this low tem­perature inactivation is usually reversible. For each enzyme there is one specific tem­perature at which its activity is at a maximum—between 25-30°G in most plant cells.

The enzymes are very sensitive to variations in pH and also to the presence of cer­tain inorganic salts. Trypsin, a proteolytic enzyme, is completely inactive in an acid pH, whereas pepsin requires an acidic medium.

The pH inactivation is, however, reversible as in the case of low temperature inactivation if the pH change is not too extreme. Most enzymes act best in a neutral or slightly alkaline medium. The activity of trypsin is at its maximum in a solution of 1% Na₂CO₃.

Catalytic Activity:

By the agency of enzymes chemical reactions readily occur in the living cells which otherwise can only be brought about, as in hydrolysis, by boiling with reagents like strong acid. A living cell cannot heat substances up in a test-tube in order to get them to break up or to combine as we do in the laboratory.

Thus the enzymes join simple molecules together to form complex molecules or break down the latter into simpler molecules at room temperature and only at atmospheric pressure.

Enzymes are very similar to in organic catalysts accelerating the rate of chemical reactions but are themselves found to be chemically unchanged at the end of the reaction.

The concentration of the enzymes affects the speed of the reaction but not the position of the equilibrium and the final quantity of product is unchanged.

As we have seen before, an enzyme transforms many times its, own weight of the substrate (the com­pound or compounds on which the enzyme acts), as molecular ratio of one to one million will easily be the normal order.

During the time the reaction is proceeding, an enzyme gradually disappears, reappearing at the end of the reaction, apparently unchanged in activity or in amount.

Enzyme Inhibitors:

Inorganic poisons may inactivate many enzymes when present in very small quan­tities; for example, cyanide (0.0001 M) inactivates some respiration enzymes. Other known inhibitors are sulphides, azides, malonate, antimycin, amytal, fluorides, iodoace- tates, etc. These inhibitors are usually specific, frequently inhibiting only one or few enzymes.

Zymogens:

Cells lacking a specific enzyme in its active form are frequently found to contain chemical precursor of the enzymes, which could be converted, under certain conditions, into the enzymes proper. Such substances were termed zymogens. The prosthetic groups combine with different proteins to constitute different enzymes.

Enzyme-Coenzyme-Activator Relationships:

Certain of the enzymes appear to consist solely of proteins. This is true, for example, of certain proteolytic enzymes which break down proteins to amino acids and also
of amylases which break down starch to glucose. But it has been known for a long time that certain enzymes require the presence of some other compound to be enzymatically active.

Thus, it appears that though some enzymes are active without any other additional component, most enzymes are enzymatically active only in presence of certain non­protein, thermostable component in addition to the protein (terms, holoenzymes and apo-enzymes have also been used to denote complete enzymes and protein components res­pectively) .

The non-protein component may be firmly attached- to the protein when it is called a prosthetic group or it may be readily removed by dialysis in which case it is called a coenzyme. In contrast to the protein portion, the coenzyme or prosthetic group is heat stable, being unaffected by boiling.

If the non-protein component is a metal ion it is sometimes called activator since the protein is inactive in absence of the metal ion. Perhaps the simplest case of this kind is enzymes in which the prosthetic group consists of a single metal ion.

The enzyme tyrosinase which attacks the amino acid tyrosine is made up of a protein portion combined with an atom of copper. If the copper atom is removed, the protein at once becomes inert. The presence of copper is essential to the functioning of these enzymes in catalysing oxidation of tyrosine.

The chemical compound on which the active enzyme acts is known as the substrate. In general, it is usually the prosthetic group or coenzyme which reacts with the substrate leading to its (substrate) breakdown.

The thiol or sulphydryl (SH) group of the protein component itself may also combine with the substrate, the starting material of a reaction. The specificity of an enzyme, however, depends on the protein component and not on the non-protein part. It is apparent that this specificity depends ultimately upon the variety of amino acids present in the protein chain, the sequence in which they are arranged and the way in which the chain forming protein is ‘folded’.

Thus several enzymes may contain the same coenzyme, but because of the difference in the protein component, each will act specifically on one substance. Tyrosinase and ascorbic acid (vitamin C) oxidase both have copper as their coenzymes yet the former acts on tyrosine (and several similar substances) and not on vitamin C; the latter acts on ascorbic acid but not on tyrosine.

There are enzymes which contain metals as activators other than copper; zinc, manganese, magnesium and iron are all known to function as non-protein activator components of various enzymes.

In case of iron the metal is not usually bound directly to the protein but is incorporated into a more complex molecule, an ironporphyrin which then becomes the prosthetic group proper.

Such ironporphyrin enzymes (cyto­chrome oxidase) include several enzymes which are essential in the chemical reactions of respiration. The iron-complex is an activator ajso of peroxidase and catalase.

The perplexing problem of the importance of micronutrients in plant growth in fantastically small amounts is getting clear now. The unquestionable importance of them in minute amounts for normal growth and development of plants is certainly because they form constituents of many essential enzymes.

The names of some specific microelement activators of enzymes have been cited before- Many enzymes possess relatively complex organic compounds as their coenzymes and in addition may also contain an activator metal ion.

Thus it would appear that many enzymes consist of three components—protein, coenzyme and activator. In absence of either the coenzyme or the metal ion, the protein component is inactive, i.e., the whole enzyme is inert. Similarly, the coenzyme or the activator is inactive in absence of the protein component.

It is a striking fact of advance of science that several vitamins first discovered as essential microfood elements for living cells, particularly in animals, are now known to function as coenzyme components of many enzymes.

This may well prove to be their sole function. In the enzymes of higher plants also, the vitamins of the B-complex group such as vitamin B₁, vitamin B₂, vitamin B₃, vitamin B₆, vitamin B₁₂, niacin, adenine, pantothenic acid, choline (an organic plant base, constituents of some essential fats, such as egg-yolk), etc., all constitute coenzyme groups.

Ascorbic acid (vitamin C) is a coenzyme of the enzyme of myrosinase which is active on a number of glucosides, particularly the mustard oil glucosides, sinigrin and glucobrassicin.

The enzyme pyruvic carboxylase, which catalyses the removal of hydrogen from the carboxyl group of pyruvic acid is a protein, diphosphothiamine magnesium (DPT), where thiamine (vita­min B₁) phosphate is the coenzyme group and magnesium is the activator.

Vitamin B₁ (thiamine or aneurine) is structurally a thiazopyrimidine compound. So the coenzyme can alternatively be called pyrimidine thiazolediphosphorie ester; the ring thus con­tains the essential element sulphur in addition to carbon and nitrogen.

The protein- coenzyme-activator ratio here is about 75,000 gm protein: 1 gm mol thiamine phosphate: 1 gm atom of Mg! The coenzymes of dehydrogenase group of enzyme include among others nicotinamide adenine dinucleotide, NAD (previously known as coenzyme I and until recently, DPN) and nicotinamide adenine dinucleotide phosphate, NADP (coenzyme II, TPN) in which both niacin and adenine appear together.

The nucleotide contains a pentose sugar, ribose. The NAD and NADP can be combined with any of the several proteins to yield a whole series of different enzymes all of which, however, remove hydrogen atoms from different oxidisable substrates.

We have seen before that when the non-protein prosthetic group of an enzyme is removable by dialysis it is called a coenzyme. The separated coenzyme is inert so also is the protein component; united again, the active enzyme is reconstituted. The protein- coenzyme relation is supposed to act like a lock and key arrangement.

The protein component of an enzyme, the lock, is inactive without the coenzyme, the key, just as a lock cannot be opened without its particular key and the key is useless without the lock. As a key fits a lock and opens the lock so does a coenzyme unite with the protein to initiate the activity of the whole enzyme.

As a particular lock has only one key which fits and opens the lock so has one particular protein component only one coenzyme for the initiation of the activity of this enzyme as a whole. Again just as one master key can open a number of locks so may one coenzyme convert a number of proteins into a series of highly active different enzymes by combining with the different proteins.

Nature of Enzyme Action:

We have discussed before that exothermal (exergonic) reactions are thermodynamically spontaneous, i.e., they are losing reactions as far as energy is concerned. But this certainly does not mean that the exergonic breakdown can, take place without any outside help.

A substance, S at energy level E, can therefore be converted to substances, X and Y, spontaneously at the lower energy level, El. Then why S does not fall of itself to energy El, at the same time splitting into substances X and Y?

The reason is that there is an energy barrier and the substance S may be too stable at energy E to roll down slowly as it were to energy level, El. In order that the substance S can go over the energy barrier it must first be activated, that is, it must be given some extra energy as for instance, such as supplied by increasing the temperature, enabling S to go uphill over the peak and for this reason the rate of reaction, i.e., S breaking into X and Y increases with rise of temperature.

But this reaction can also be made to take place without any rise in temperature, in the presence of highly reactive substances acting in extremely small quantities and known as catalyst—inorganic or organic.

If the re­action involves two substances, the enzyme may accelerate it by adsorbing these sub­stances on its surface, bringing them closer, and thereby reducing the energy barrier. When a gas stove is turned on, the gas does not burn until and unless the activation energy is supplied by a lighted match stick.

The enzyme certainly forms an unstable intermediate compound with the subs­tance, S. The energy barrier may thus be lowered to such a value that the energy of the moving molecules in the solution can now easily overcome the barrier and the substance S, falling to the lower energy level El, breaks down into subs targes X and Y with the release of exergonic energy

S + enzyme → enzyme. S enzyme. S –>X + Y + enzyme +energy.

The net result is, therefore, that the catalyst has permitted the reaction to take place by removing the so-called energy gap. It is evident that the catalyst may be active in minute quantities because it is regenerated after each reaction.

Classification of Enzymes:

The classification of enzymes is generally based on the nature of the reactions they catalyse rather on the nature of the enzyme itself, since in so many cases, the exact chemical nature is still largely unknown.

The terminology of the enzymes is often arbitrary; the modern tendency is to name them after substrate or the nature of re­action, adding the Greek suffix -ase. Certain classical names such as pepsin, trypsin, bromelin, are, however, still retained.

Enzymes are classified into six classes:

(1) Oxidoreductases;

(2) Transferases;

(3) Hydrolases;

(4) Lyases;

(5) Isomerases; and

(6) Ligases.

They are further sub­divided according to the groups within the substrate, which they attack, modify or transfer, the bonds- broken down or formed, the nature of acceptors etc.

The Inter­national Union of Biochemistry has given each enzyme a number which gives a general idea regarding the nature of its action. Thus, alcohol dehydrase which catalyses the reaction.

CH₃CH₂OH + NAD ↔ CH₃GHO + NADH + H⁺ is given the number 1.1.1.1 because it is a dehydrogenase (Class 1), it acts on the CHOH group (Subclass 1), it was NAD as the acceptor (Sub-Subclass 1) and it is the first enzyme in the Sub-Subclass.

We shall now discuss very briefly some major types of enzyme reactions with a few illustrations.

(A) Hydrolytic and Condensation Reactions (Hydrolases):

In most cases hydrolysis results in a large release of energy (exothermal reactions) while in condensation, energy is absorbed (endothermal reactions). Hydrolysis always involves use, of at least one molecule of water.

These reactions can be represented as follows:

A.B is a substance which splits into A.H. and B.OH in presence of the enzyme. Enzyme usually accelerates the hydrolysis, If, however, the substance A.B. can be removed as rapidly as it is formed, the enzyme can also accelerate the condensation reaction, at least in some cases. These are called hydrolases.

Most of the complex organic substances in plants are stable in aqueous solutions but are readily split into their constituents if the necessary enzyme is present. The hydrolytic enzymes are subdivided into four groups.

The presence of water is essential for the chemical reactions to take place:

The most important esterase is lipase which controls the hydrolysis of fats into fatty acids and glycerol. Other esterases are phosphatases, chlorophyllases, etc.

The carbohydrases catalyse the hydrolysis of the glycosidic bonds in carbohydrates to set free the constituent sugars. Invertase controls the hydrolytic splitting of sucrose (cane sugar) into glucose and fructose.

Other carbohydrates and glycosides (compounds of carbohydrates with one or more non-sugar components), are also hydrolysed by these enzymes, e.g., amylase {diastase), cellulase, maltose, myrosinase, etc. In every case, the carbohydrates are ultimately split into the simplest hexoses or pentoses.

(3) Proteolytic Enzymes:

These enzymes accelerate the splitting of complex protein molecules into proteoses, peptones, polypeptides and ultimately into amino acids. The most important proteolytic enzymes are trypsin, pepsin, papain, bromelin, etc.

Papain, we know now, consists of 202 amino acids, the sequence and the order of which is now known almost completely. Papain is a sulphydril protease of approx. molecular weight, 23,000.

(4) Amidases:

Asparaginase splits the amide asparagine into asparagine and ammonia.

(B) Transfer Reactions (Transferases):

In these reactions enzymes catalyse the transfer of radicals from one compound to the other:

AB + C → C.B. + A

These reactions usually result in small energy changes and hence are readily reversible. Enzymes of this group accelerate both synthesis and breakdown reactions. These are called transferases.

Many chemical groups, e.g., CH₅, CH₂OH-CHO, glycosyl, nucleotidyl, etc. can be transferred by these enzymes from one compound to another. Transaminases transfer amino groups; transphosphatases transfer the phosphate group (transfers PO₄ group from H₃PO₄ to starch, etc.).

(C) Oxidation-Reduction Reactions (Oxidoreductases):

In this type of reactions enzymes themselves or some other compounds act as hydrogen acceptors in the breakdown of one of the substances concerned, getting reduced in the process:

The substance is oxidised by the removal of hydrogen which (hydrogen) is taken up by the enzyme (coenzyme), getting reduced in the process.

The reduced coenzyme may in turn be oxidised by another hydrogen acceptor and (e.g., another enzyme) thus freed again to take part in the reaction:

BH₂ + enzyme ↔ B +enzyme.H₂

These are the most heterogeneous of all the three classes. Zymase, the first known of all the enzymes, falls into this class. Zymase is now known to be a complex of several enzymes, all of which catalyse either oxidation or reduction reactions in plant and animal cells.

Carboxylase, one of the previously known components of zymase complex which splits off CO₂ from carboxyl groups, can be compared to a dehydrogenase for the reaction is really a dehydrogenation of the carboxyl group into acetaldehyde.

As we have seen before, the most important enzymes of this class are those controlling the transfer of hydrogen atoms from one compound to another, when one compound is oxidised and the other reduced. These are known as dehydrogenases. The enzyme, actually the coenzyme, may itself act as a hydrogen acceptor;

For example:

Other well-known dehydrogenase coenzymes are FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide); FAD acts specifically as electron or hydrogen ion carrier between the nicodnamide coenzymes (NAD or NADP) and the ubiquinone, coenzyme £).

In the above reaction alcohol is oxidised, i.e., dehydrogenated by the removal of hydrogen atom to aldehyde and the NAD is reduced. The reduced NADH can in turn be oxidised by another hydrogen acceptor. If the NADH or any other similarly reduced enzyme is leoxidised not by removal of hydrogen but by molecular oxygen, it is called oxidase.

AH₂ + enzyme → A + enzyme. H₂

enzyme. H₂ +1/2 HO₂ →-enzyme+H₂O

One of the most important of these oxidase enzyme-complexes arc the various cytochromes and cytochrome oxidases.

Two other well-known oxidation-reduction enzymes are peroxidases and catalases; both of these control the decomposition of H₂O₂ or organic peroxide. When a peroxidase acts on peroxide, inorganic or organic, the molecular oxygen thus released at once oxidises another compound.

When catalase acts on a peroxide, free molecular oxygen is released. The presence of catalase in plant tissue can easily be demonstrated if a block of potato tissue is dropped in a test-tube containing H₂O₂; the evolution of oxygen can at once be seen.

The metabolic significance of the universal presence of catalase in plant or animal cells is not clear. It is possible that catalase acts as a safety-valve in preventing accumulation of peroxides, produced as a result of cell metabolism.

(D) Carboxylase is a Lyase Type of Enzyme which Catalyses Reversible Reactions:

The lyases snap e—C, C—O, G—N and G—S bonds, without the agency of water. Isocitrate lyase converts isocitrate into succinate and glyoxalate; phhospophyruvate carboxy­lase degrades the substrate oxalacetate in presence of inorganic phosphate to phosphoenol pyruvate, CO₂ or H₂O.

The isomerases isomerise the same or different molecules, e.g., alanine recemasc, ribulose-phosphate 3-epinaerase, Triosephosphate isomerase, etc.

The ligases form G—O, G—S or G—N bonds. They are responsible for syntheses of many aminoacyl-tRNA, acyl coenzyme AS, asparagine, glutamine, nucleic acids and proteins.

Distribution of Enzymes in the Cell:

From all observations as yet available it appears that the enzymes which the plant cells contain are usually localised in the protoplasm. One of the most amazing facts about enzymes is the huge number of different kinds that are certainly located in the extremely small space occupied by protoplasm of a single mature cell.

Some enzymes are present as the soluble component of the protoplasm whereas many other enzymes are bound firmly into the structure of various particles which are always found in protoplasm. Numerous enzymes, as for example phosphorylase, are found to be functionally associated with protoplasm.

Other enzymes are found attached to the microscopic particles of protoplasm such as mitochondria (minute, 1-1/4 µ, semi-solid bodies found in the protoplasm of all cells except bacteria and blue-green algae), or the plastids.

The number of enzymes in a cell is large. The orderly way in which the series of metabolic reactions take place in a cell, points to the distinct possibility that the orga­nisation of the enzymes in a cell is according to a pattern evolved for the functioning of the cell as a living entity.