CHAPTER V.

ACTION OF ENZYMES.

“The living organism is enabled by the use of enzymes to bring about, under ordinary conditions of temperature and moderate concentrations of acid or alkali, many chemical reactions which would otherwise require a high temperature or powerful reagents.”--W. M. BAYLISS.

In a recent work,[94] Dr. Bayliss defines enzymes as the “catalysts produced by living organisms.” A catalyst is a body which greatly accelerates the rate of reaction in chemical processes, without apparently taking part in the process. For instance, peroxide of hydrogen is decomposed into oxygen and water by mere contact with finely divided platinum, while the latter remains unaltered in the process. In this case the platinum black is the catalyst.[95]

[94] “The Nature of Enzyme Action,” by Dr. W. M. Bayliss, FRS.

[95] The important discoveries of MM. Paul Sabatier and Senderens on the catalytic action of finely divided metals, notably nickel and copper, have recently been extended to a study of the catalytic action of various metallic oxides. In the _Comptes rendus_, MM. Paul Sabatier and A. Mailhe give an account of a new synthetic method, based on the catalytic effect of titanium oxide, which would appear to possess many practical applications. They show that if a column of titanium dioxide is maintained at a temperature of 280°–300° C., and a mixture of the vapours of a primary alcohol and a fatty acid (other than formic acid) is led over it, the corresponding ester is formed. The same limit is here reached instantaneously as was found by Berthelot after prolonged contact. An excess of either constituent favours the limit of combination of the other. Following this method, the methyl, ethyl, propyl, butyl, isobutyl, and isoamyl esters of acetic, propionic, butyric, isobutyric, isovaleric and caproic acids have been prepared. Esters of benzyl alcohol have also been readily obtained by this method. The inverse action--the direct hydrolysis of esters by water--is also easily effected, and the use of titanium dioxide reduces any secondary reactions to a negligible amount. See Nature, March 9, 1911, p. 54. See also Dr. Sand’s paper--bibliography.

In natural processes the best known type of an enzyme is diastase (amylase), the enzyme contained in malt, and which enables the malt to convert starch into dextrin and sugar (maltose). It is capable of transforming more than 2000 times its own weight into sugar, which fact is quite sufficient to show that its action differs from that of an ordinary chemical reaction. Another enzyme, sucrase, according to O’Sullivan and Thompson, will hydrolyze 100,000 times its weight of cane sugar to invert sugar. Rennet will coagulate 250,000 times its own weight of casein in milk. The list of enzymes grows longer almost daily, as some new one is separated having a specific action, until one is almost led to believe that the mechanism of life itself, as manifested in the cell, is due to enzymes.

It has been found that enzymes act very much in the same way as inorganic catalysers. As an example, the velocity of the reaction of invertase (the enzyme of yeast which hydrolyses cane sugar to grape sugar) has been compared with the same hydrolysis brought about by heating a solution of cane sugar with a mineral acid. In both cases the reaction is in accordance with the law of mass action (Guldberg and Waage) that the amount of sugar transformed will decrease as less remains to be transformed. In the diagram (Fig. 26) the curve A is for invertase (Jas. O’Sullivan, “Journ. Inst. of Brewing,” vol. v. p. 168); curve B is for the hydrolysis by acid (Wilhelmy), from which it will be seen that the manner in which the hydrolysis proceeds is practically the same in both cases.[96]

[96] There are apparent exceptions and complications of this law which we shall not here enter into, except to say that they may be explained by the fact that the action of some enzymes is reversible (see p. 141, under lipase.)

In the case of fermentation by the living organism, the fermentation rises rapidly, and then gradually slows down and comes to an end before the whole of the fermentable matter is used up; the curve, therefore, is of a hyperbolic character, C in the diagram, which represents, in a general way, the fermentation of glucose by B. furfuris. The ordinates represent the amount of acid produced by the bacteria. The time in this case would be more nearly represented by hours instead of minutes on the abscissa.

The mathematical expression for the velocity of the reaction is

_dx_/_dt_ = _k_(_a_ − _x_)

where

_a_ = original concentration of solution

_x_ = the quantity transformed in time t

_k_ = coefficient of velocity of the reaction

By integrating the above equation, it may be shown that

1/_t_ log(_a_/(_a_ − _x_)) = _k_

For the experiment of J. O’Sullivan with invertase, _k_ has a mean value of 0·0013; for Wilhelmy’s experiment with acid, _k_ has a mean value of 0·001377.

Enzymes are produced by the living cell, with other secretions, and as a consequence are found in all plants and animals. Certain organs, however, produce enzymes in large quantities, or appear to be specially set apart for their production. In plants, the seeds are the chief seat of enzyme activity; in animals, certain glands, such as the salivary glands and the pancreas. The mucous membrane of the stomach and intestines also secrete enormous quantities.

The production of enzymes by bacteria was observed by Wortmann in 1882. It has been found that the secretion of the enzymes depends upon the composition of the nutrient medium in which the bacteria are grown. For instance, Pfeffer found that the secretion of diastase by Bacillus megatherium depended upon the amount of cane sugar in the nutrient medium. The cane sugar checked the secretion of the diastase, and the same effect was observed in the case of the common mould Penicillium glaucum. In Bacillus mesentericus vulgatus, diastase has been found to exist side by side with four other enzymes. Passini,[97] in studying the putrefactive anaerobic bacteria of the normal human intestines, succeeded in separating from B. putrificus a proteolytic enzyme filtered free from bacteria, and which caused proteolysis in media which were too acid to permit of the bacteria growing. The enzyme easily dissolved, without previous neutralization, the coagulated casein caused in milk by old coli cultures. Acid gelatin media were also liquefied by the enzyme.

[97] “Zeit. f. Hygiene,” Bd. xlix. p. 135.

From the evidence we have at present it seems probable that every variety of enzyme, hydrolysing, oxidizing, ammoniacal, etc., can be produced by bacteria.

The following useful classification of enzymes is due to Effront.[98]

[98] Les Enzymes, Dr. Jean Effront, Paris, 1899.

----------------------------+-------------------+--------------------- Name of Enzyme |Substance on which | Products of the | the Enzyme acts | Reaction ----------------------------+-------------------+--------------------- A. HYDROLYZING ENZYMES | | | | 1. _Fermenting Carbo- | | hydrates_: | | Sucrase or invertin |Cane sugar |Invert sugar Diastase or amylase |Starch and dextrin |Maltose Maltase or glucase |Dextrin and maltose|Glucose Lactase |Milk sugar |Glucose and galactose Trehalase |Trehalose |Glucose Inulase |Inulin |Levulose Cytase |Cellulose |Sugars Pectase |Pectin |Pectates and sugars Caroubinase |Carobin |Carobinose | | 2. _Fermenting Glucosides_:| | Emulsin |Amygdalin and |Glucose, oil of | other glucosides | bitter almonds, | | and prussic acid Myrosin |Myronate of potash |Glucose and allyl | | isosulphocyanate Betulase |Gaultherin |Oil of gaultheria, | | glucose Rhamnase |Xanthoramin |Rhamnetine, | | isodulcite | | 3. _Fermenting Fats_ | | (Lipolytic): | | Steapsin |Fats } |Glycerin and Lipase |Fats } | fatty acids | | | | 4. _Fermenting Proteids_: | | Rennet |Casein |Caseuin Plasmase |Fibrinogen |Fibrin Casease |Casein { | Pepsin |Albuminoids { |Proteoses, peptones Trypsin} |Ditto { |Proteose, peptones, Papain } |Ditto { | amides | | 5. _Fermenting Urea_: | | Urease |Urea |Ammonium carbonate | | B. OXIDIZING FERMENTS | | Laccase |Uruschic acid, |Oxyuruschic acid, | tannin, anilin, | various oxidation | etc. | products Oxydin |Colouring matters |Ditto | of cereals | Malase |Ditto, of fruits |Ditto Olease |Olive oil |Ditto Tyrosinase |Tyrosin |Ditto Oenoxydase |Colouring matter |Ditto | of wine | | | C. FERMENT WHICH SPLITS | | UP THE MOLECULE | | Zymase |Various sugars |Alcohol and | | carbon-dioxide ----------------------------+-------------------+---------------------

A more recent classification based on chemical properties is that of Kossel and Dakin.[99] They divide ferments into two classes:--

(1) Oxylytic ferments capable of breaking the O-link by which the radicals are held together in fats and carbohydrates.

(2) Imino-lytic ferments, including the amino-lytic ferments which act on the amino groups of urea.

Group 2 is sub-divided into--

(_a_) Trypsin and erepsin, which separate the imide NH from the neighbouring carbonyl CO.

(_b_) Arginase, which separates off urea from arginin.

[99] Zeit. f. Physiol. Chem. xli. f. 153 (1904). See also the articles by Mr. A. Seymour Jones, B.Sc., writing under the pseudonym of “Heof Joppa,” in the Leather Trades’ Review, July 19, 1911, p. 540; and Aug. 16, 1911, p. 625.

Although it is somewhat doubtful whether the enzymes contained in dog dung are of glandular origin,[100] it is quite certain that other enzymes are secreted by bacteria developing in the dung while it is kept prior to being used for puering. These enzymes may be separated by the following method. About 150 c.c. of puer is well mixed with an equal quantity of glycerin, and allowed to stand for seven days. It is then filtered through paper by means of a pump, and yields a clear filtrate of a deep golden-brown colour; the filtrate is poured in a thin stream into a tall vessel containing about 1500 c.c. of 98 per cent. alcohol. A copious flocculent precipitate of the albuminous matter and enzymes is thrown down, the solution is filtered and the precipitate washed on the filter with absolute alcohol, and then dried over sulphuric acid in vacuo. The resulting powder is, of course, a mixture of all the albumins in solution, and probably only a small portion of it consists of the pure enzymes. We have merely succeeded by this method in concentrating them. The property of albuminous bodies in the act of coagulation to carry down soluble matter is well known, and this also renders the preparation of any pure proteid extremely difficult. It may be mentioned here that recent evidence goes to show that enzymes are not of a proteid nature, since, by repeated purification, the proteid matter may be almost entirely got rid of, while the activity of the residue containing the enzyme becomes considerably greater.

[100] See, however, the recent paper by Eberle and Krall, Ueber den Nachweis des Trypsins im Hundekot, Collegium 1911, p. 201, in which the authors endeavour to show the presence of unchanged trypsin in dog dung. Their proof depends upon the action of an antipancreatic serum on infusions of dog dung based on the work of Achalme, Ann. de l’Inst. Pasteur, 1901, p. 737. See also the criticism on this paper by Dr. Otto Röhm and Dr. Max Goldman, Collegium, 1911, p. 265. Hammarsten (Physiol. Chemistry, 1911, p. 494) states that “among the secretions which undergo putrefaction in the intestine, the pancreatic juice, which putrefies most readily, takes place first.”

Krawkov’s method of preparing diastase from saliva (Green, p. 46) may also be mentioned, as it is of general application. It consists in salting out the enzymes, by saturating the clear solution (in this case saliva diluted with an equal volume of water) with neutral ammonium sulphate. The precipitate which is caused by the saturation is collected on a filter, and washed for a short time with strong alcohol. It is then allowed to stand under absolute alcohol for one or two days, and finally dried at 30° C. On extraction with water it yields a solution which is strongly diastatic, and which gives no proteid reactions.

There are several other methods for the preparation and purification of enzymes, but up to the present it may safely be stated that no one has succeeded in preparing an enzyme in a state of purity.

In considering the mechanism by which enzymes act, it must be remembered in the first place that they are colloids, and, as such, will form absorption compounds with the substrate, or body, upon which they are acting. It is difficult to understand how an enzyme can exert an action on the substrate, unless it enters into some kind of combination with it, although this may be only a temporary one. The action of some enzymes has been found to be due to extremely small amounts of certain metals, e.g. in the case of the oxidizing enzyme laccase, the metal is manganese. In the purest samples of this enzyme prepared, 0·16 per cent. Mn was found, and it has been supposed by some observers that the whole of the action of the enzyme may be attributed to the _physical state_ of the manganese which it contains. According to this hypothesis,[101] the active part of the enzyme is the ion Mn. This ion may exist in the solution in two conditions, differing by the electric charge which they carry. One of them Mn_{++} carries two positive charges, the other Mn_{+++} carries three. In the first phase, Mn_{++} is transformed into Mn_{+++} by absorbing the charge of one ion of hydrogen (H_{+}), and two hydrogen ions thus discharged, in the _nascent state_, unite with the oxygen dissolved in the liquid to form water.

[101] Duclaux. See Bibliography.

2Mn_{++} + 2H_{+} + 0 = 2Mn_{+++} + H_{2}O

In the second phase the ion Mn_{+++} with three charges will be transformed into the ion with two charges, by decomposing a molecule of water, of which the nascent oxygen will attach itself to the oxidizable body R yielding the oxide RO.

2Mn_{+++} + H_{2}O + R = 2Mn_{++} + 2H_{+} + RO

and the same cycle of operations will begin again, and continue indefinitely.

In the above illustration the enzyme action was an oxidizing one. In the case of puering it is a hydrolytic action, in other words a molecule of water is added to the skin substance. The fibre or some portion of it is converted first into proteoses, and finally into peptones, and simpler bodies. The active metal in these cases appears to be calcium, but by what mechanism it brings about the hydrolysis is at present unknown.

Pozerski[102] found that the pancreatic juice, which is secreted after injections of certain sera (anti-pancreatic action), and as a consequence has no pancreatic action, contains no calcium, but pancreatic juice secreted under the influence of pilo-carpin is more or less rich in calcium, and its proteolytic action increases about equally with the amount of calcium contained in it. The same probably holds good for the intestinal juice.

[102] Koch’s Jahresbericht über Gärhungs Organismen, 1908, 635.

Victor Henri has shown that the power of metals in the colloidal state to bring about these catalytic actions varies with the metal employed, and is in inverse ratio to the size of the particle. There is a very interesting and wide field of research open here in order to determine the conditions under which the various metals act. To this end the ashes from the purest enzyme preparations might be studied, and methods devised for producing these metals in the colloidal state, for it seems evident that it is the _state_ of the body acting which gives it the properties observed, and not its chemical properties in the usual sense.

The enzymes contained in dog dung which are effective in the puering process belong to several groups, principally the proteolytic and lipolytic groups, but indirectly enzymes of the first group (see p. 132), (fermenting carbohydrates) and of the fifth group (fermenting urea) also play some part by decomposing various compounds (e.g. cellulose and urea) contained in the puer.

The action of certain enzymes from the animal body upon skin has been tried by the author.[103] Those selected were pepsin and pancreatin,[104] as being most likely to be present in dung. Pepsin only acts in presence of HCl. Two portions of the same skin were taken, one of them was treated with a 1 per cent. solution of pepsin, acidified with 0·2 per cent. of hydrochloric acid; the other in a bate liquor of dogs’ dung (puer), both at a temperature of 40°C. At the end of one hour the skin in the pepsin solution was considerably “fallen,” but that in the manure solution was bated nearly away, i.e. the greater part of it was dissolved. A 1 per cent. solution of pancreatin (Mercks) was found to act far more rapidly than pepsin; 1·5 per cent. of chloroform was added to the solution, to prevent the development of bacteria. The skin was reduced, but had not the peculiar touch of a puered skin. As will be shown later, this was found to be due to the absence of any chemical action upon the lime salts in the skin, and consequently it felt “limey.” This action took place in the puered skin, but not in the skin treated with pancreatin alone.

[103] J.S.C.I., vol. xiii., 1894, p. 218.

[104] The word pancreatin is used throughout in the sense of pancreas extract. As is well known, this contains several enzymes, trypsin, steapsin, maltase, and also a rennin.

W. J. Salomon (9) has also attributed the activity of the bate to pepsin and pancreatin, but he does not give any proof of the presence of these ferments in the bate.

Since it is practically impossible up to the present, to separate these enzymes from the dung in a state of purity, the method described on p. 134 was employed.

The enzymes prepared in this way consist of a mixture of all the enzymes present in the dung, the amount obtained from 1000 grm. of dung being about 4 grm. The product had a slight diastatic action upon starch; 0·5 grm. in 100 c.c. of water at 35° C. was found to have a very considerable reducing action upon skin, and when combined with the amine compounds prepared from the dung, the action was more powerful, and more rapid than with puer. Limed skin was puered in thirty minutes in this solution to a perfect condition, in the absence of bacteria, and with no evil smelling compounds. The reaction of the solution at the beginning of the experiment was faintly alkaline; at the end of the experiment it was considerably alkaline.

This experiment proves that the action of the dung is a complex one, due to the combined action of enzymes and chemical compounds upon the skin. These compounds, which are principally amines, and salts of amino-acids, probably assist the enzymes, and at the same time act upon the lime remaining in the skin from the previous liming process. Whether a skin, which has never been submitted to the liming operation, could be bated by enzymes alone, without the addition of amines, has not, so far as I know, been tried, but it is highly probable that this would be the case.

In order to compare the action of the enzymes prepared from dung with that of the enzymes produced by bacteria, a mixed culture of bacteria from puer in dextrose gelatin, after seven days’ growth, was taken. 200 c.c. of this was mixed with 200 c.c. of dilute alcohol (65 per cent.), and well shaken: gelatin and albuminoid bodies are by this means precipitated. The liquid was filtered and poured into eight times its volume of 98 per cent. alcohol. The precipitate which came down was washed with absolute alcohol, and dried in vacuo. The enzymes thus obtained were re-dissolved in water, and the former experiment with the skin repeated with this solution, with the addition of the amines. The skin was brought down in exactly the same way as before, showing conclusively that it is the enzymes produced by bacteria, acting in conjunction with the amines, which bate the skin. It would seem that the action of the enzymes is aided by the presence of amine compounds, in addition to the chemical action which these latter have upon the skin. The action is interdependent, i.e. bacterial action alone is insufficient, and chemical action alone is insufficient, the true bating action being a combination of the two.

There seems little doubt that it is the enzymes which dissolve the skin substance, or rather certain parts of the intercellular substance of the fibres, and the compound of this substance with lime. The action is a digestive one, and may be compared, as we have shown, to that of the digestive ferment of the pancreas.

This fact has been made use of in the artificial bate “Oropon” (see