CHAPTER XIV
ENZYMES AND THEIR ACTION
The characteristic difference between the reactions of inorganic compounds and those of organic substances lies in the rapidity, or velocity, of the chemical changes involved. Speaking generally chemical reactions take place between substances which are in solution, so that they may come into sufficiently intimate contact that chemical action between them can take place. There are, of course, occasional examples of reactions between dry solids, such as the explosion of gunpowder, etc., but the general rule is that reacting materials must be in either colloidal or true solutions.
Inorganic materials, when dissolved in water, usually ionize very readily. That is, they are not only disintegrated into individual _molecules_, but a considerable proportion of these molecules separate into their constituent _ions_. When solutions containing ionized compounds are brought together, conditions for chemical interaction are ideal, and the reaction proceeds with such tremendous rapidity as to be completed almost instantaneously, in most cases.
Organic compounds, on the other hand, ionize only very slowly, if at all. Hence, reactions between organic compounds, even when they are in solution, proceed very slowly unless carried on at high temperatures, under increased pressure, or under the influence of some catalytic agent. Even under the stimulation of these reaction-accelerating agencies, most chemical changes in organic compounds when carried on in the laboratory, require several hours or even days and sometimes weeks, for their completion. But when similar reactions take place in living organisms, they proceed with velocities which resemble those of inorganic compounds in the laboratory. This difference between the velocity of organic reactions when carried on under artificial conditions in the laboratory (often spoken of as "_in vitro_") as compared with that of the same reactions when they take place in a living organism ("_in vivo_"), is due to the universal presence in the living protoplasm of certain organic catalysts, known as _enzymes_.
ENZYMES AS CATALYSTS
The phenomenon known as "catalysis" is of common occurrence in both inorganic and organic chemistry. The effect of a small amount of manganese dioxide in aiding in the liberation of oxygen from potassium chlorate is an example which is familiar to all students of elementary chemistry. Similarly, spongy platinum accelerates the oxidation of sulfur dioxide to sulfur trioxide, in the commercial manufacture of sulfuric acid. Again, the hydrolysis of sucrose into fructose and glucose proceeds very slowly in the presence of water alone, but if a little hydrochloric acid or sulfuric acid be added to the solution, the velocity of the hydrolysis is enormously accelerated. Many other examples of the accelerating effect of various chemicals upon reactions into which they do not themselves enter, might be cited.
The essential features of all such catalytic actions are: (1) the velocity of the reaction is greatly altered, usually accelerated; (2) the catalytic agent does not appear as one of the initial substances, or end-products, of the reaction, and is not itself altered by the chemical change which is taking place; (3) the accelerating effect is directly proportional to the amount of the catalyst which is present; (4) relatively small amounts of the catalyst produce very large results in the reacting mixture; and (5) the catalysts cannot themselves initiate reactions, but only influence the velocity of reactions which would otherwise take place at a different rate (usually much more slowly) in the absence of any catalytic agent.
Enzymes conform to all of these properties of catalysts, and are commonly defined as the "catalysts of living matter." They are almost universally present in living organs of every kind, and perform exceedingly important functions, both in the building-up of synthetic materials and in the rendering soluble of the food of both plants and animals, so that it can be translocated from place to place through the tissues of the organism.
Enzymes differ from inorganic catalysts in being destroyed by heat, in not always carrying the reaction to the same stage as does the inorganic catalyst which may accelerate the same reaction, and in producing different changes in the same substance by different enzymes.
The name "enzyme" comes from Greek words meaning "in yeast," as the nature and effect of the enzyme involved in the alcoholic fermentation of sugars by yeast were those which were first recognized and understood. It was at first thought, by Pasteur and his students, that fermentation is the direct result of the life activities of the yeast plant. Later, it was found that water extracts from sprouted barley, from almond seeds, and from the stomach, pancreas, etc., were able to bring about the decomposition of starch, of amygdalin, and of proteins, respectively, in a way which seemed to be quite comparable to the fermentative action of yeasts. Hence, it was thought that there were two varieties of active agents of this kind, one composed of living cells and the other non-living chemical compounds, and these were called the "organized ferments" and the "unorganized ferments," respectively. However, in 1897, Büchner found that by grinding yeast cells with sharp sand until they were completely disintegrated and then submitting the mass to hydraulic pressure, he could obtain a clear liquid, entirely free from living cells, which was just as active in producing fermentation as was the yeast itself. This discovery paved the way for a long series of investigations, which have conclusively demonstrated that there is no distinction between "organized" and "unorganized" ferments, that all living organisms perform their characteristic functions by means of the enzymes which they contain, and that these enzymes can bring about their characteristic catalytic effects outside the cell, or tissue which elaborates them, just as well as within it, provided only that the conditions of temperature, acidity or alkalinity of the medium, etc., are suitable for the particular enzyme action which is under consideration.
GENERAL PROPERTIES OF ENZYMES
Since enzymes are catalysts, it is plain that an accurate description of their activity should, in each case, refer to the influence which they exert upon some definite reaction velocity. But since the phrases necessary to describe such an effect are cumbersome and inconvenient, and since most of the reactions which are accelerated by the catalytic action of enzymes are either simple hydrolyses, changes in oxygen content, or other simple decompositions or condensations, which will otherwise proceed so slowly as to be practically negligible, it is customary to speak of the enzyme as "acting upon" the material in question. It should be understood, however, that this is a misstatement, as the enzyme cannot actually initiate a reaction, or "act upon" any substance; it only acts as a catalyzer to accelerate the action of water, oxygen, etc., upon the material in question.
Generally speaking, most enzymes are colloidal in form and, hence, do not diffuse through membranes such as the cell-walls. Some of them perform their characteristic functions only within the cell, or organ, which elaborates them, and can be obtained outside these tissues for purposes of study only by first rupturing the cell-wall or other membrane with which they are surrounded. Such enzymes are known as "intracellular." Others are regularly secreted by glands which discharge them onto other organs, as the stomach or intestines of animals, where they perform their useful functions; or, as in the case of germinating seeds, they move to other parts of the organ, and can be extracted from the tissue by simple treatment with water. These are known as the "extracellular" enzymes.
Enzymes are specific in their action. Any given enzyme affects only a single reaction; or at most acts only upon a single group of compounds which have similar molecular configuration. Usually it is only a single compound whose decomposition is accelerated by the action of a particular enzyme; but there are a few enzymes, such as _maltase_ (which acts on all [alpha]-glucosides) and emulsin (which acts on all [beta]-glucosides) which act catalytically upon groups of considerable numbers of similar compounds.
Enzymes, like all other catalysts, act more energetically at increased temperatures; but for each particular enzyme there is an "optimum temperature," (usually between 40° and 65°) above which the destructive effect of the temperature upon the enzyme itself more than offsets the accelerating influence of the increased temperature. At still higher temperatures (usually 80° to 100°) the enzymes are "killed," i.e., rendered permanently inactive. All enzymes are "killed" by boiling the solutions in which they are contained. Dry preparations of enzyme material can withstand somewhat higher temperatures, for somewhat longer periods of time, than can the same enzyme in moist condition or in solution. When an enzyme has once been inactivated by heating, or "killed," it can never be restored to activity again.
Enzymes are extremely sensitive to acids, bases, or salts, their activity being often enormously enhanced or, in other cases, entirely inhibited, by the presence in the reacting medium of very small amounts of free acids, or bases, or even of certain neutral salts. For example, pepsin, the enzyme of the stomach will act only in the presence of a slightly acid medium and is wholly inactive in a mixture which contains even the slightest amount of free alkaline material; while trypsin, the similar enzyme of the intestine, acts only under alkaline conditions. Practically all enzymes are rendered inactive, but not destroyed, by the presence of either acid or alkali in excess of N/10 strength. Many will act only in the presence of small quantities of certain specific neutral salts; while, on the other hand, other salts are powerful inhibitors of enzyme action. Enzymes often differ from the protoplasm which secretes them in their response to antiseptics, such as toluene, xylene, etc., which inhibit the activity or growth of the cell, but have no effect upon the activity of the enzymes which it contains.
THE CHEMICAL NATURE OF ENZYMES
Nothing is known with certainty concerning the chemical nature of enzymes. Being colloidal in nature, they adsorb carbohydrates, proteins, fats, etc., so that active enzyme preparations often respond to the characteristic tests for these groups of substances; and many investigators have reported what has, at first, seemed to be conclusive evidence that some particular enzyme which they have studied is either a carbohydrate, a protein, or some other type of organic compound. Later investigations have always shown, however, that if the preparation in question be submitted to the digestive action of the enzymes which hydrolyze the particular type of substances to which it is supposed to belong, the material will lose its characteristic protein, or carbohydrate, etc., properties, without losing its specific activity, thus clearly indicating that the substance which responds to the characteristic tests for some well-known type of organic compounds is present as an impurity and is not the enzyme itself.
The present state of knowledge concerning the nature of enzymes seems to indicate that, like the inorganic catalysts, they may vary widely in chemical composition; and that their tremendous catalytic effects are due, in part at least, to their colloidal nature. This will be better understood and appreciated after the phenomena associated with the colloidal condition have been considered (see the following chapter).
NOMENCLATURE AND CLASSIFICATION
Since nothing is known of the chemical composition of enzymes, they can only be studied by considering the effects which they produce. This is reflected in the systems which have been adopted for their nomenclature and classification.
As they were first supposed to be proteins, the earlier representatives of the group were given characteristic names ending with the suffix _in_, similar to that of the proteins. Since this idea has been found to be incorrect, however, a system of nomenclature has been adopted which assigns to each enzyme the name of the material upon which it acts, followed by the suffix _ase_. Thus, cellulase is the enzyme which accelerates the hydrolysis of cellulose; glucase, that acting upon glucose; amylase, that acting upon starch (_amylum_), etc.
The substance upon which the enzyme acts (or, strictly speaking, the substance whose hydrolysis, oxidation, or other chemical change, is catalytically affected by the enzyme) is called the _substrate_.
Most enzymes are catalysts for hydrolysis reactions and are, hence, classed as _hydrolytic_ in their action, and may be spoken of as "hydrolases." Those which accelerate oxidation are called "oxidases"; while those that stimulate reduction reactions are "reductases"; those that aid in the splitting off of ammonia, or amino-acid groups, are "deaminases"; and those that aid in the splitting off of CO_{2} from COOH groups are "carboxylases," etc.
The hydrolytic enzymes are further subdivided into the sucroclastic (sugar-splitting), or sucrases; the lipoclastic (fat-splitting), or lipases; the esterases (ester-splitting); proteoclastic (protein-splitting), or proteases; etc.
OCCURRENCE AND PREPARATION FOR STUDY
Enzymes are present in all living matter. In animal tissues, they occur in the largest amounts in those glands or organs where active vital processes take place, as in the brain, the digestive tract, blood, etc. In plants, they may be found in all living cells, and are especially abundant in the seeds, where they serve to render soluble and available to the young plant the stored food materials. The enzymes of moulds, and other parasitic plants, are usually extracellular in type, being secreted for the purpose of making the material of the host plant available to the parasite. Extracellular enzymes are also developed in seeds during germination, in order that the stored food material of the endosperm may be rendered soluble and translocated into the tissues of the growing seedling. But most other plant enzymes are intracellular in type. Hence, in all preparations of plant enzymes for study, or for commercial use, the first step in the process is, necessarily, a thorough rupturing of the cell-walls of the plant material.
The rupturing of the cells may be accomplished in a variety of ways, as follows: (1) mechanical disintegration, as by grinding in a mortar with sharp sand; (2) freezing the material, by treatment with liquid air, then grinding; (3) killing the cells by drying, by treatment with alcohol or acetone, then grinding the mass in a paint mill with toluene; (4) killing the cells by chemicals (sulfuric acid, 0.5 to 1.0 per cent, or other suitable agents) followed by extraction with water; (5) autolysis, or self-digestion, in which the cells are mixed with toluene or some other antiseptic which kills the cells without injuring the enzymes, then the material is minced or ground up and suspended in water containing the antiseptic, until the enzymes dissolve the cell-walls and so escape into the liquid--this process being especially adapted to the preparation of active extracts from yeasts, which contain the necessary cell-wall dissolving enzymes to facilitate autolysis.
Enzymes may be separated out of the aqueous extract obtained from cells ruptured by any of the above methods, by precipitation with alcohol, acetone, or ether, in which they are insoluble; but if this is done, the precipitate must be at once filtered off and rapidly washed and dried, as prolonged contact with these precipitating agents greatly diminishes the activity of most enzymes. Or, they may be adsorbed out of solution on gelatinous, or colloidal, materials, like aluminium hydroxide, or various hydrated clays. If the dry preparations obtained in any of these ways are contaminated by carbohydrates, proteins, etc., these may be removed by treatment with suitable digesting enzymes obtained from the saliva, gastric, and pancreatic juices, and the digested impurities washed out with 60 to 80 per cent alcohol, leaving the enzyme preparation in a purified but still active form.
In any study of the "strength," or possible catalytic effects, of an enzyme preparation, it is necessary, first, to determine what particular reaction it affects, by qualitative tests with various substrate materials, such as starch, sugars, glucosides, proteins, etc., and then to determine quantitatively its accelerating effect upon the reaction in question. The latter may be done by measuring either the _time_ required to carry a unit quantity of the substrate material through any determined stage of chemical change, or the _quantity_ of the substrate which is changed in a unit period of time. It would not be profitable to go into a detailed discussion here of the methods of making these quantitative measurements of enzyme activity. Such discussions must necessarily be left to special treatises on methods of study of enzyme action. It may be said, however, that generally both the qualitative tests for, and the quantitative measurements of, the accelerating influence of enzymes depend upon the observation of some change in the physical properties of the substrate material, such as the optical activity, electrical conductivity, or viscosity, of its solution. In some cases, it is convenient to make an actual quantitative determination of the amount of end-products produced in a given time, as in the inversion of cane sugar, the hydrolysis of maltose, etc., but such determinations necessarily involve the removal of some of the reaction mixture for the purposes of the determinations, and are not, therefore, suitable for the study of the progressive development of the reaction which is being studied.
Enzymes are found in all parts of the animal organism and those which are active in the digestion of food, the metabolism of digested material, the coagulation of blood, etc., have been extensively studied. A discussion of these animal enzymes would be out of place in such a text as this, however, and the following list includes only enzymes which are known to occur in plant tissues. These well-known enzymes will serve as examples of the several general types which have thus far been isolated and studied.
------------------- -+-----------+-------------+-------------+----------- Class and Type. | Enzyme. | Substrate. |End-products.| Found in. ---------------------+-----------+-------------+-------------+----------- I. Hydrolases | | | | (_a_) Esterases |Lipase |Fats |Glycerol and |Oily seeds | | |fatty acids | | | | | (_b_) Carbohydrases |Sucrase or |Sucrose |Glucose and |Yeasts |invertase | |fructose | | | | | |Maltase |Maltose and |Glucose, etc.|Barley malt | |all [alpha]- | | | |glucosides | | | | | | |Dextrinase |Dextrin |Maltose |Malt | | | | |Inulase |Inulin |Fructose |Artichokes, | | | |etc. | | | | |Amylase or |Starch |Maltose |Malt, etc. |diastase | | | | | | | |Cellulase |Cellulose |Maltose |Bacteria | | | | |Pectinase |Pectose |Arabinose |Fruits | | | | |Cytase |Hemi- |Mono- |Nuts, | |celluloses |saccharides |seeds, etc. | | | | (_c_) Glucosidases |Emulsin |Amygdalin |Glucose, |Almond | |and all |etc. |kernels, | |[beta]- | |etc. | |glucosides | | | | | | |Maltase |All [alpha]- |Glucose, |Barley malt | |glucosides |etc. | | | | | |Myrosin |Sulfur- |Glucose, |Mustard | |containing |etc. |seeds | |glucosides | | | | | | |Rhamnase |Xanthorhamnin|Rhamnose, |_Rhamnus | | |etc. |spp._ | | | | |Phytase |Phytin |Inosite and |Bran coats | | |H_{3}PO_{4} |of seeds | | | | (_d_) Proteases |Erepsin |Proteins |Amino-acids |Many plants | | | | |Papain |Proteins |Amino-acids |Papaws | | | | |Bromelin |Proteins |Amino-acids |Many plants | | | | |Nuclease |Nucleo- |Proteins and |Many plants | |proteins |nucleic acid | | | | | II. Oxidases | | | | (_a_) Catalases | ........ |Hydrogen |Water and |Nearly | |peroxide |oxygen |all plants | | | | (_b_) Peroxidases | ........ |Organic |"Active" |Nearly | |peroxides |oxygen |all plants | | | | (_c_) Oxidases | ........ |Chromogens |Pigments |Many plants | | | | | |Alcohols and |Acids |Many plants | |phenols | | (_d_) Reductases | ........ | ......... | ......... |Many plants | | | | III. Deaminases |Urease |Urea |Ammonia and | | | |CO_{2} | | | | | |Guanase |Guanine |Xanthine | | | | | |Adenase |Adenine |Hypoxanthine | | | | | IV. Carboxylases | | | | (_a_) | ........ |Keto-acids |Aldehydes | | | |and CO_{2} | | | | | (_b_) | ........ |Amino-acids |Amines and | | | |CO_{2} | | | | | V. Coagulation |Pectase |Coagulates | .......... |Fruits enzymes | |pectic | | | |bodies | | | | | | VI. Fermentation |Zymase |Glucose, etc.|Alcohol and |Yeasts enzymes | | |CO_{2} | | | | | |Lactic acid|Fatty acids |Lactic acid |Bacteria |ferment | | | | | | | |Butyric |Fatty acids |Butyric acid |Bacteria |acid | | | |ferment | | | | | | | ---------------------+-----------+-------------+-------------+----------
The above list includes only the more common and best-known plant enzymes. It seems reasonable to suppose that for every individual type of organic compound which may occur in general plant groups, or even in single species, there is a corresponding enzyme available to affect its physiological alterations. Indeed, new preparations of active enzymes from special types of plants and new evidences of the existence of enzymes in various plant organisms are continuously being reported.
A few of the most common specific representatives of individual groups of enzymes may be briefly described, as follows:
=Amylase= (or =diastase=, as it was first named and is still commonly called) is probably the most widely distributed enzyme of plants. It is found in practically all bacteria and fungi; in practically all seeds (it has been found in active form in seeds which were known to be over fifty years old); in all roots and tubers; and in practically all leaves, where it is located in the stroma of the chloroplasts.
It appears to exist in two modifications, known, respectively, as (_a_) translocation diastase and (_b_) diastase of secretion. The first form is found in the cells of ungerminated seeds, in leaves, shoots, etc. It remains in the cells where reserve starch is stored and aids in the transformation of starch into soluble materials for translocation from cell to cell. It is active at a lower temperature than the second form, its optimum temperature being 45° to 50°. The second form is secreted by the scutellum, and perhaps by the aleurone cells, of germinating seeds, being produced by special glandular tissue. It aids in the hydrolysis of the starch for the use of the growing embryo. Its optimum temperature is 50° to 55°.
The activity of amylase is accelerated by the presence of small quantities of neutral salts, especially by sodium chloride and disodium phosphate. It acts best in neutral solutions, its activity being inhibited, although the enzyme itself is not destroyed, by the presence of more than minute traces of free mineral acid or alkali.
=Sucrase= (or invertase) is present in almost all species of yeasts, where it serves to convert unfermentable sucrose into glucose and fructose, which are readily fermentable. Invertase is also present in moulds and other microorganisms; and in the buds, leaves, flowers, and rootlets of those higher order plants which store their carbohydrate reserves in the form of sucrose. It appears that sucrose, while easily soluble, is not readily translocated, or utilized, by plants until after it has been hydrolyzed into its constituent hexoses.
The optimum temperature for invertase is 50° to 54°; it is killed if heated, in the moist condition, to 70°. Its activity is increased by the presence of small amounts of free acids; but is inhibited by free alkalies.
=Zymase= is the active alcoholic fermentation enzyme of yeasts. It accelerates the well-known reaction for the conversion of hexose sugars into alcohol and carbon dioxide, namely,
C_{6}H_{12}O_{6} = 2C_{2}H_{5}OH + 2CO_{2}.
Because of its scientific interest and industrial importance in the fermentation industries, its action has been extensively studied. It acts only in the presence of soluble phosphates and of a coenzyme (see below) which is dialyzable and not destroyed, which is probably an organic ester of phosphoric acid. The significance of the molecular configuration of the hexose sugars in their susceptibility to action by zymase has already been discussed in detail (see page 56).
The optimum temperature for zymase action is 28° to 30°. The enzyme is killed by heating to 45° to 50° in solution, or to 85° if in dry preparation.
=Proteases= of the erepsin type, i.e., those which break proteins down to amino-acids instead of only to the proteose or peptone stage, as is characteristic of the enzymes of the trypsin type, are widely distributed in plants. Except in the case of the two which occur in large amounts in certain special fruits (papain in papaws, and bromelin in pineapples), they are very difficult to prepare in pure form for study. In general, all proteolytic actions, even when accelerated by active enzymes, proceed much more slowly than do the hydrolyses of carbohydrates or fats. It seems that metabolic changes of the complex protein molecules are much more difficult to bring about and take place much more slowly than do those of the energy-producing types of compounds.
The presence of proteolytic enzymes in most vegetative cells, and in seeds, may be demonstrated, however, by studying the action of extracts of these tissues upon soluble proteins. The best-known example of this type of enzymes is the protease of yeast; but similar ones may be found in germinating seeds. These vegetable proteases are usually most active in neutral or only faintly alkaline solutions, and their activity is nearly always inhibited by even traces of free acids.
Most laboratory studies of proteolytic enzymes are carried on with preparations of the powerful members of this class of enzymes which are found in the digestive tract of animals, namely, the pepsin of the gastric juice, which acts in the acid medium, in the stomach, and the trypsin of the pancreatic juice, which acts in the alkaline medium of the intestinal tract. But even these powerful proteases require several hours for the transformation of an amount of soluble albumin into its amino-acid constituents which is equivalent to the amount of starch which is hydrolyzed to maltose by diastase in a very few minutes.
Enzymes which govern oxidative changes, known respectively, as _catalases_ and _oxidases_, are almost universally present in plants. Catalase decomposes peroxides, with the liberation of free oxygen. It is, therefore, necessary to the final step in the process of photosynthesis, as elucidated by Usher and Priestley (see page 26), and serves to prevent the destructive action of hydrogen peroxide upon chlorophyll. The almost universal presence of oxidases in plant tissues has been repeatedly demonstrated. They are present in especially large amounts in tissues which are being acted upon by parasitic fungi or are combating unfavorable conditions of growth. The oxidases, in such cases, seem to be the agents by which the plant is able to stimulate its metabolic activities to overcome the unfavorable environment for its normal development.
In vegetables and fruits, the common browning, or blackening, of the tissues when cut surfaces are exposed to the air has been demonstrated to be due to the catalytic oxidation of the tannins or of certain amino-acids, especially tyrosine, under the influence of the oxidases which are present in the tissues. In fact, most pigmentation phenomena are due to changes in the oxygen content of the chromogens of the cells of the plant, under the influence of the oxidases which are present in the protoplasm of the cells in question. Hence, the oxidases may be said to be the controlling agencies for both the energy-absorbing activities and for respiration in plants.
THE NATURE OF ENZYME ACTION
The mechanism by which an enzyme accomplishes its catalytic effects has been the object of extensive studies during recent years, especially since the discovery by Büchner that enzymes could be isolated in solutions entirely free from the disturbing influence of growing cells. Several theories concerning the mode of this catalytic action have been advanced. The earliest and simplest of these was that the enzyme simply creates an environment favorable for the particular chemical reaction to take place, as by exposing large surfaces of the substance in question to the action of the hydrolytic, or other effective, agent, by means of surface adsorption of the substrate material on the colloidal enzyme.
However, more recent investigations clearly indicate that there is an actual combination between the substrate material and the enzyme, which combination then breaks down with a resultant change in the substrate material and a freeing of the enzyme for repeated recombination with additional substrate, with the net result that the chemical change in the substrate material is enormously accelerated. That such a combination between substrate and enzyme actually exists has been demonstrated in two different ways: (_a_) experimentally, by mixing together solutions of an enzyme and of its substrate, each of which is filterable through paper or through a porous clay filter, with the result that the active material in the combined solutions will not pass through these same filters; and (_b_) mathematically, by a study of the curves representing the reaction velocities of typical reactions which are proceeding under the influence of an enzyme, which show that so long as there is a large excess of substrate material present, the accelerating influence of the catalyst is uniform over given successive periods of time, but that when the quantity of substrate material becomes smaller than that which permits the maximum combining power of the enzyme to be exercised, the reaction velocity immediately slows up.
Again, the fact that the specificity of the action of an enzyme, i.e., the limitation of the action of that enzyme to a specific single compound or group of similar compounds, is definitely related to the molecular configuration of the molecule of the substrate, as has been found to be true in all those cases where the molecular configuration of the substrate material has been established (see pages 56 to 58), is an added indication that there is some kind of a union between the enzyme and the substrate as a first step in the catalytic process.
As to the nature of this supposed combination of substrate and enzyme, two theories are held. The first is that this union is in the form of an actual molecular combination, or chemical compound, and the other is that it is a purely physical, or colloidal complex. The latter view has by far the greater weight of theoretical and experimental evidence in its support. The relation of electrolytes to the catalytic effect of enzymes, the appearance of the reacting masses under the ultramicroscope, and the effect of heat upon the reacting mixtures, all point to the conclusion that the phenomenon is colloidal rather than molecular in character. This view also makes the remarkable catalytic effects which take place in living protoplasm, which undoubtedly exists in the colloidal condition, much more easily understood. This phase of the matter will be much more apparent after the chapter dealing with the physical chemistry of the protoplasm has been studied.
A further indication that the mechanism of enzyme activity is colloidal in character lies in the fact that, so far as is known, all reactions which are catalyzed by specific enzymes are reversible and the same enzyme will accelerate the velocity of the reaction in either direction, the direction in which the reaction goes being determined by the conditions surrounding the reacting material at the time. It was formerly supposed that enzymes catalyze only decomposition reactions and that the synthetic reactions of living tissues are produced by means of some other force or agency. This view supported the idea of a chemical union of the enzyme with the substrate which, when it breaks down, breaks the molecule of the substrate material into some simpler form, or forms. But it is now known that the reaction which is influenced by the enzyme will be catalyzed in either direction by the specific enzyme which "fits" the particular substrate material at every point of its molecular configuration, as the glove fits the hand. The contrast between this fitting of the enzyme to the entire configuration of the molecule, and the union at a single point or group which is characteristic of chemical linkages, is apparent. As examples of the synthetic action of the same enzyme which, under other conditions, accelerates the decomposition of the same material, there may be cited the demonstrated synthesis of isomaltose from glucose by maltase; the production of ethyl butyrate from alcohol and butyric acid; and the synthetic production of artificial fats, by the aid of the pancreatic lipase; and the apparent synthesis of a protein from the same amino-acids which may be obtained from it by hydrolysis under the influence of the same protease, but under different environmental conditions.
ACTIVATORS AND INHIBITORS
The activity of enzymes is strongly influenced by the presence in the solution of other bodies, usually, although not always, electrolytes. This is probably due, in most cases at least, to the action of the electrolyte upon the colloidal condition of the enzyme. All enzymes do not respond alike to the action of the same electrolyte, however. The activity of certain enzymes is enormously increased by the presence of a small amount of acid; while the action of another may be absolutely inhibited by the same acid in the same concentration. Thus, the activity of the amylase found in the endosperm of many seeds is instantly stopped by adding to the solution enough sulfuric acid to make it two-hundredth normal in strength; while the same concentration of acid actually accelerates the activity of some of the proteases.
Formaldehyde, hydrocyanic acid, and soluble fluorides usually inhibit both the activity of a cell and of the enzymes which it contains; while other antiseptics, such as toluene, xylene, etc., prevent the growth of the cell, or organism, without interfering with the activity of the enzymes which may be present. By the use of this latter type of antiseptics, it is possible to distinguish between chemical changes which are involved in the actual development of a cell and those which can be brought about in other media by means of the enzymes which are contained in the cell.
Any substance which increases the catalytic activity of an enzyme is known as an "accelerator," or "activator"; while one which prevents this activity is called an "inhibitor," or "paralyzer."
A type of accelerating influence quite different from that of electrolytes is found in the effect of certain amino-acids upon enzyme action. The influence of small amounts of asparagine in enormously increasing the hydrolytic effect of amylase is an example. There is no known explanation for this type of activation of the enzyme.
The influence of activators, or inhibitors, in providing favorable or unfavorable conditions for the action of an enzyme, should not be confused with the relation to the enzyme itself of what are known as "coenzymes" and "antienzymes," discussed in the following paragraph.
COENZYMES AND ANTIENZYMES
In the cases of many enzymes of animal tissues, it has been found that they are absolutely inactive unless accompanied by some other substance which is normally present in the gland, or protoplasm, which secretes them. Thus, the bile salts are absolutely necessary to the activity of trypsin, in its characteristic protein-splitting action. Such substances are known as "coenzymes." They can usually be separated from their corresponding enzymes by dialysis, the coenzyme passing through the parchment membrane. Such coenzymes are not killed by boiling the dialyzate, and the activity of the enzyme is restored by adding the boiled dialyzate to the liquid which remains within the dialyzer.
The best known example of a coenzyme in plant tissues is in connection with the activity of the zymase of yeast cells. If yeast juice be filtered through a gelatin filter, the colloidal enzymes which are left behind are entirely inactive in producing fermentation, but may be restored to activity again by mixing with the filtrate. An examination of this filtrate, which contains the coenzyme for zymase, shows that it contains soluble phosphates and some other substance whose exact nature has not yet been determined, both of which are necessary to the activity of the zymase. The phosphates seem to enter into some definite chemical combination with the substrate sugars, while the other coenzyme seems to be necessary in order to make possible the final breaking down of the sugar-phosphate complex by the zymase. This phenomenon of coenzyme relationship is not very frequently observed in plant enzyme studies, probably because the coenzyme (if there be such, in the case which is under observation) usually accompanies the enzyme itself through the various processes of extraction and purification of the material for study. However, care must be taken in all cases when dialysis is employed, to see that a possible coenzyme is not separated from an otherwise active preparation.
An entirely different type of phenomenon is that exhibited by "antienzymes." These are found in the various intestinal worms which live in the digestive tracts of animals; and prevent the digestive action of the enzymes of the stomach and intestines upon these worms. Probably similar "antienzymes" are located in the mucous linings of the intestinal tract itself, and serve to prevent the auto-digestion of these organs by the active enzymes with which they are almost continually in contact.
The difference between an antienzyme, which protects material which would otherwise be subject to the attack of an enzyme, and an inhibitor, which renders the enzyme itself inactive, is apparent.
So far as is known, however, no such substances as antienzymes are present in plant tissues; although the question as to why the proteoclastic enzymes which are elaborated by a given mass of protoplasm do not attack the protoplasm itself, might well be raised.
ZYMOGENS
It is apparent that, since enzymes are produced by protoplasm for the special needs of any given moment or stage of development, there must be a preliminary stage, or condition, in which they do not exert their characteristic catalytic effect. When in this stage, the compound is known as "proenzyme," or "zymogen." In this stage, it is inactive, but can be made to exhibit its catalytic effect, usually by bringing it into contact with a suitable activator. When once so activated, however, it cannot be returned again to the inactive state.
This phenomenon has been studied in connection with the zymogens of the digestive proteases, pepsin and trypsin. Trypsinogen may be rendered active by contact with either calcium salts or with another substance (apparently itself an enzyme) known as enterokinase, which is secreted in the intestinal tract.
Similarly, proenzymes have been reported as occurring in numerous plant tissues. These proenzymes are believed to be present in the plant cells in the form of definite characteristic granules, which may be observed under the microscope, and which disappear when the enzyme becomes active. Thus, "proinulase" has been reported as occurring in artichoke tubers: "prolipase," in castor beans; "proinvertase," in several species of fungi; and, probably, "prooxidase," in tobacco leaves. In the case of the last-named zymogen, it has been observed that after the zymogen has been once activated, as in response to the need for increased activity due to the entrance of the germs of certain leaf-diseases, it can once again produce a second supply of the enzyme, but the process cannot again be repeated.
Calcium salts, or very dilute acids, are usually energetic activators of proenzymes.
PHYSIOLOGICAL USES OF ENZYMES
There can be no doubt that enzymes exert a tremendously important influence in vital phenomena, by determining the rate at which the chemical changes which are involved in these phenomena shall proceed. Since they do not initiate reactions, and since they may catalyze reversible reactions in either direction, it cannot be said that they determine the type of reactions which will take place in any given mass of protoplasm; but, undoubtedly, they do exert a determining influence upon the rate at which the reaction will proceed, after the protoplasmic activity has determined the direction in which it shall go.
Without the intervention of these catalyzing agents, it would be impossible for reactions between these non-ionized organic components of the cell contents to come to completion with anything like the marvelous rapidity with which these changes must take place in order to permit the organism to grow, to perform its necessary vital functions, or to adjust itself to the changes in its environmental conditions.
Since the number of different reactions which take place within a living cell is very great, and since these chemical changes are extremely variable in type, it follows that the number of different enzymes which must exist in either a plant or an animal organism is likewise very large. For example, fourteen different enzymes have been isolated from the digestive system, and at least sixteen from the liver, of animals. They are universally present in living protoplasm of every kind, from the most minute bacterium to the largest forest trees, in the plant kingdom; and from the am[oe]ba to the whale, in animals.
While there is a great variety of enzymes which may be produced by a single individual organism, the same enzyme may be found in the greatest variety of organisms; as, for example, the protease trypsin, which has been found in several species of bacteria, in the carnivorous plant known as "Venus' Fly Trap," and in the human pancreas, as well as that of all other animals.
FURTHER STUDIES NEEDED
From the discussions which have been presented in this chapter, it is apparent that the enzymes play a tremendously important part in vital phenomena, by controlling the rate at which the biochemical reactions take place in the cells of the living organism.
The means by which the protoplasm elaborates these all-important chemical compounds are as yet absolutely unknown. Even the nature of the enzymes themselves is still a matter of speculation and study. Much intensive study is needed and should be given to these matters, for the purpose of elucidating the methods by which the enzymes accomplish their remarkable catalytic effects, and, if possible, the actual chemical nature of the enzymes themselves. It is conceivable, of course, that if the latter object of these studies should ever be reached, it might be possible to synthetize enzymes artificially, and so to develop a means for the artificial duplication of the synthesis of organic compounds with the same velocity that this is done in the plant cells. Such a result would have a scientific interest fully as great as did Wöhler's artificial synthesis of urea, which proved that there is no essential difference in character between the compounds which are the products of living organisms and those which are produced in the laboratory; and, at the same time, might have an immensely more important practical bearing, since it would lead the way to the artificial production of the carbohydrates, proteins, fats, etc., for which we are now dependent upon plant growth as the source of these materials for use as human food.
References
BAYLISS, W. M.--"The Nature of Enzyme Action," 186 pages, _Monographs_ on Biochemistry, London, 1919 (4th ed.).
EULER, H., trans. by POPE, T. H.--"General Chemistry of the Enzymes," 319 pages, 7 figs., New York, 1912.
EFFRONT, J., trans. by PRESCOTT, S. C.--"Enzymes and their Application,--Enzymes of the Carbohydrates," 335 pages, New York, 1902.
EFFRONT, J., trans. by PRESCOTT, S. C.--"Biochemical Catalysts in Life and Industry--Proteolytic Enzymes," 763 pages, New York, 1917.
GREEN, J. R.--"The Soluble Ferments and Fermentation," 512 pages, Cambridge, 1901, (2d ed.).
GRUS, J.--"Biologie und Kapillaranalyse der Enzyme," 227 pages, 58 figs., 3 plates, Berlin, 1912.
HARDEN, A.--"Alcoholic Fermentation," 156 pages, 8 figs., Monographs on Biochemistry, London, 1914.
PLIMMER, R. H. A.--"The Chemical Changes and Products Resulting from Fermentations," 184 pages, London, 1903.
OPPENHEIMER, C., trans. by MITCHELL, C. A.--"Ferments and their Actions," 343 pages, London, 1901.