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:
maltose + maltase ⇆ glucose + glucose + maltase.
fat + lipase ⇆ glycerin + fatty acid + lipase.
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 _amylase_[16] (starch-splitting enzyme);
_invertase_,[16] which splits cane-sugar into dextrose and levulose:
C₁₂H₂₂O₁₁ + H₂O = C₆H₁₂O₆ + C₆H₁₂O₆. _Lipase_[16] or a fat-splitting
enzyme, which decomposes fat into glycerin and fatty acid:
C₃H₅(OC{n}H₂{n}₋₁O)₃ + 3H₂O = C₃H₅(OH)₃ + 3C{n}H₂{n}O₂.
Fat Glycerin 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.)