The Fundamentals of Bacteriology

CHAPTER XII.
PHYSIOLOGICAL ACTIVITIES (Continued).

PRODUCTION OF ENZYMES.

Most of the physiological activities of bacteria which have been discussed are due to the action of these peculiar substances, so that a knowledge of their properties is essential. This knowledge cannot as yet be exact because no enzyme has, up to the present, been obtained in a “pure state,” though it must be admitted that there are no certain criteria which will enable this “pure state” to be recognized. It was formerly thought that they were protein in nature, but very “pure” and active enzymes have been prepared which did not give the characteristic protein reactions, so this idea must be abandoned. That they are large moleculed colloidal substances closely related to the proteins in many respects must still be maintained. There are certain characteristics which belong to enzymes, though no one of them exclusively. These may be enumerated as follows:

1. Enzymes are dead organic chemical substances.

Dead is used in the sense of non-living, never having lived, not in the sense of “ceased to be alive.”

2. They are always produced by living cells:

Sometimes as active enzymes, sometimes as pro-enzymes or zymogens which are converted into enzymes outside the cell by acids, other inorganic substances or other enzymes.

3. They produce very great chemical changes without themselves being appreciably affected.

Enzymes will not continue to act indefinitely, but are used up in the process (combination with products?). The amount of change is so great in proportion to the amount of enzyme that the above statement is justified in the relative sense. Thus a milk-curdling enzyme has been prepared that would precipitate 100,000,000 times its own weight of caseinogen.

4. Their action is specific in that each enzyme acts on one kind of chemical substance only, and the products are always the same.

The substance may be combined with a variety of other chemical substances so that the action appears to be on several, but in reality it is on a definite group of molecules in each instance. For example, emulsin attacks several different glucosides but always sets free dextrose from them.

5. The action is inhibited and eventually stopped, and in some cases the enzyme is destroyed by an accumulation of the products of the action. If the products are removed, the action will continue, if the enzyme is not destroyed. This effect is explained partly because the enzyme probably combines with some of the products, since it does not act indefinitely, and partly because of the reversibility of the reaction.

6. Like many chemical reactions those of enzymes are reversible, that is, the substance broken up may be reformed by it from the products produced in many instances. Thus:

7. The presence of certain mineral salts seems to be essential for their action. These and other substances which are necessary are sometimes called co-enzymes. A salt of calcium is most favorable for a great many.

8. They may be adsorbed like other colloids by “shaking out” with finely divided suspensions like charcoal or kaolin, or by other colloids like aluminum hydroxide or proteins.

9. When properly introduced into the tissues or blood of an animal, they cause the body cells to form anti-enzymes which will prevent the action of the enzyme (see Chapter XXVII).

10. Though inert, they show many of the characteristics of living organisms, that is

(a) Each enzyme has an optimum, a maximum and a minimum temperature for its action.

All chemical reactions have such temperature limits, the distinction is that for enzymes as for living substance the range is relatively narrow.

(b) High temperatures destroy enzymes. All in water are destroyed by boiling in time and most at temperatures considerably below the boiling-point. When dry, many will withstand a higher degree of heat than 100° before they are destroyed.

(c) Temperatures below the minimum stop their action, though they are not destroyed by cold.

(d) Many poisons and chemical disinfectants (Chapter XIV) which kill living organisms will also stop the action of enzymes, though generally more of the substance is required, so that it is possible to destroy the living cells by such means and yet the action of the enzyme will continue.

(e) Most enzymes have an optimum reaction of medium either acid, alkaline or neutral, depending on the particular enzyme, though some few seem to act equally well within a considerable range on either side of the neutral point.

The final test for an enzyme is the chemical change it brings about in the specific substance acted on.

The most prominent characteristic of enzymes is that they bring about very great chemical changes without themselves being appreciably affected. This property is also shown by many inorganic substances which are spoken of as “catalytic agents” or “catalyzers” so that enzymes are sometimes called “organic catalyzers.” The function of catalytic agents seems to be to hasten the rate of a reaction which would occur spontaneously, though in a great many cases with extreme slowness.

Just how enzymes act is not certain and probably will not be until their composition and constitution are known. Most probably they form a combination with the substance acted on (the substrate) as a result of which there is a rearrangement of the atoms in such a way that new compounds are formed, nearly always at least two, and the enzyme is at the same time set free. It is rather remarkable that chiefly optically active substances are split up by enzymes and where two modifications exist it is usually the dextro-rotatory one which is attacked. No single enzyme attacks both. This probably means that the structure of the enzyme corresponds to that of the substrate, “fits it as a key fits a lock,” as Emil Fischer says.

The production of enzymes is by no means restricted to bacteria since all kinds of living cells that have been investigated have been shown to produce them and presumably all living cells do. Hence the number of different kinds of enzymes and of substances acted upon is practically unlimited. Nevertheless they may be grouped into a comparatively few classes based on the general character of the change brought about by them.

I. Class I is the so-called “splitting” enzymes whose action is for the most part hydrolytic, that is, the substance takes up water and then splits into compounds that were apparently constituents of the original molecule. As examples may be mentioned diastase, the enzyme first discovered, which changes starch into a malt-sugar, hence is more commonly called amylase16 (starch-splitting enzyme); invertase,16 which splits cane-sugar into dextrose and levulose: C₁₂H₂₂O₁₁ + H₂O = C₆H₁₂O₆ + C₆H₁₂O₆. Lipase16 or a fat-splitting enzyme, which decomposes fat into glycerin and fatty acid:

Proteases, which split up proteins into proteoses and peptones.

Other classes of “splitting enzymes” break up the products of complex protein decomposition, such as proteoses, peptones and amino-acids. A variety of the “splitting enzymes” is the group of

“Coagulases” or coagulating enzymes as the rennet (lab, chymosin) which curdles milk; fibrin ferment (thrombin, thrombase) which causes the coagulation of blood. These apparently act by splitting up a substance in the fluids mentioned, after which splitting one of the new products formed combines with other compounds present (usually a mineral salt, and in the cases mentioned a calcium salt) to form an insoluble compound, the curd or coagulum.

Another variety is the “activating” enzymes or “kinases” such as the enterokinase of the intestine. The action here is a splitting of the zymogen or mother substance or form in which the enzyme is built up by the cell so as to liberate the active enzyme.

Of a character quite distinct, from the splitting enzymes are

II. The zymases. Their action seems to be to cause a “shifting on rearrangement of the carbon atoms” so that new compounds are formed which are not assumed to have been constituents of the original molecule. Most commonly there is a closer combination of the carbon and oxygen atoms, frequently even the formation of CO₂ so that considerable energy is thus liberated. Examples are the zymase or alcoholase of yeast which converts sugar into alcohol and carbon dioxide; C₆H₁₂O₆ = 2C₂H₆O + 2CO₂: also urease, which causes the change of urea into ammonia and carbon dioxide. Another common zymase is the lactacidase in lactic acid fermentation.

III. Oxidizing enzymes also play an important part in many of the activities of higher plants and animals. Among the bacteria this action is illustrated by the formation of nitrites, nitrates and sulphates and the oxidation of alcohol to acetic acid as already described.

IV. Reducing enzymes occur in many of the dentrifying bacteria and in those which liberate H₂S from sulphates. A very widely distributed reducing enzyme is “catalase” which decomposes hydrogen peroxide.

As previously stated, most of the physiological activities of bacteria are due to the enzymes that they produce. It is evident that for action to occur on substances which do not diffuse into the bacterial cell—starches, cellulose, complex proteins, gelatin—the enzymes must pass out of the bacterium and consequently may be found in the surrounding medium. Substances like sugars, peptones, alcohol, which are readily diffusible, may be acted on by enzymes retained within the cell body. In the former case the enzymes are spoken of as extra-cellular or “exo-enzymes,” and in the latter as intra-cellular or “endo-enzymes.” The endo-enzymes and doubtless also the exo-enzymes may after the death of the cell digest the contents to a greater or less extent and thus furnish substances that are not otherwise obtainable. This process of “self-digestion” is known technically as “autolysis.”

A distinction was formerly made between “organized” and “unorganized ferments.” The former term was applied to the minute living organisms, bacteria, yeasts, molds, etc., which bring about characteristic fermentative changes, while the latter term was restricted to enzymes as just described. Since investigation has shown that the changes ascribed to the “organized ferments” are really due to their enzymes, and that enzymes are probably formed by all living cells, the distinction is scarcely necessary at present.

PRODUCTION OF TOXINS.

The injurious effects of pathogenic bacteria are due in large part to the action of these substances, which in many respects bear a close relationship to enzymes. The chemical composition is unknown since no toxin has been prepared “pure” as yet. It was formerly thought that they were protein in character, but very pure toxins have been prepared which failed to show the characteristic protein reactions. It is well established that they are complex substances, of rather large molecule and are precipitated by many of the reagents which precipitate proteins. Toxins will be further discussed in Chapter XXVII. It will be sufficient at this point to enumerate their chief peculiarities in order to show their marked resemblance to enzymes.

1. Toxins are dead organic chemical substances.

2. They are always produced by living cells.

3. They are active poisons in very small quantities.17

4. Their action is specific in that each toxin acts on a particular kind of cell. The fact that a so-called toxin acts on several different kinds of cells, possibly indicates a mixture of several toxins, or action on the same substance in the cells.

5. Toxins are very sensitive to the action of injurious agencies such as heat, light, etc., and in about the same measure that enzymes are, though as a rule they are somewhat more sensitive or “labile.”

6. Toxins apparently have maxima, optima, and minima of temperature for their action, as shown by the destructive effect of heat and by the fact that a frog injected with tetanus toxin and kept at 20° shows no indication of poison, but if the temperature is raised to 37°, symptoms of poisoning are soon apparent. Cold, however, does not destroy a toxin.

7. When properly introduced into the tissues of animals they cause the body cells to form antitoxins (Chapter XXVII) which are capable of preventing the action of the toxin in question.

8. The determining test for a toxin is its action on a living cell.

It is true that enzymes are toxic, as are also various foreign proteins, when injected into an animal, but in much larger doses than are toxins.

A marked difference between enzymes and toxins is that the former may bring about a very great chemical change and still may be recovered from the mixture of substances acted on and produced, while the toxin seems to be permanently used up in its toxic action and cannot be so recovered. Toxins seem very much like enzymes whose action is restricted to living cells.

Just as enzymes are probably produced by all kinds of cells and not by bacteria alone, so toxins are produced by other organisms. Among toxins which have been carefully studied are ricin, the poison of the castor oil plant (Ricinus communis); abrin of the jequirity bean (Abrus precatorius); robin of the common locust (Robinia pseudacacia); poisons of spiders, scorpions, bees, fish, snakes and salamanders.

It has been stated that some enzymes are thrown out from the cell and others are retained within the cell. The same is true of toxins, hence we speak of exo-toxins or toxins excreted from, and endo-toxins or toxins retained within the cell. Among the pathogenic bacteria there are very few which secrete toxins when growing outside the body. Clostridium tetani or lockjaw bacillus, Corynebacterium diphtheriæ or the diphtheria bacillus, Clostridium botulinum or a bacillus causing a type of food poisoning, Pseudomonas pyocyanea or the blue pus bacillus are the most important. Other pathogenic bacteria do not secrete their toxins under the above conditions, but only give them up when the cell is disintegrated either within or outside the body. For the reason that endotoxins are therefore difficult to obtain, their characteristics have not been much studied. The description of toxins as above given is intended to apply to the exo-toxins of bacteria, sometimes spoken of as true toxins, and to the vegetable toxins (phytotoxins) which resemble them.

The snake venoms and probably most of the animal toxins (zoötoxins) are very different substances. (See Chapter XXIX.)

CAUSATION OF DISEASE.

This subject belongs properly in special pathogenic bacteriology. It will be sufficient to indicate that bacteria may cause disease in one or more of the following ways: (a) blocking circulatory vessels, either blood or lymph, directly or indirectly; (b) destruction of tissue; (c) production of non-specific poisons (ptomaines, bases, nitrites, acids, gases, etc.); (d) production of specific poisons (toxins).

ANTIBODY FORMATION.

Bacteria cause the formation of specific “antibodies” when properly introduced into animals. This must be considered as a physiological activity since it is by means of substances produced within the bacterial cell that the body cells of animals are stimulated to form antibodies. (See Chapters XXVI–XXIX.)

STAINING.

The reaction of bacteria to various stains is dependent on their physico-chemical structure and hence is a result of physiological processes, but is best discussed separately (Chapter XIX).

CULTURAL CHARACTERISTICS.

The same is true of the appearance and growth on different culture media. (Chapter XX.)