Enzymes

ENZYMES (syn. enzymes; French diastases), biological agents that catalyze most chemical reactions underlying the life activity of the cell and organism. A number of characteristic properties—thermolability, specificity of action, high catalytic efficiency, colloidal nature, and complex, in most cases still not elucidated chemical structure—in their aggregate allow to more or less clearly demarcate enzymes from other non-biological catalysts. The first fermentative processes known to humanity since time immemorial are fermentation (see). The very name "enzyme" derives from Latin fermentatio and denotes an agent causing fermentation. Considerably later attention was drawn to another category of fermentative processes, not possessing the characteristic violent course and gas formation of fermentation—these are the hydrolysis (see) processes of complex organic compounds—proteins, fats, carbohydrates. The dissolution of proteins by gastric juice was described by Spallanzani in 1783, and the saccharification of starch by malt by Kirchhoff in 1815. Only after the works of Berzelius and Mitscherlich, who created the concept of catalysis (see) and catalysts, did it become possible to speak of enzymes as certain chemical substances of biological origin possessing the typical properties of catalysts. The first step toward isolating such a substance in at least partially isolated state was the preparation obtained by Payen and Persoz (1833) that saccharified starch—enzyme from malt (so-called diastase), by precipitating an extract of malt with alcohol. The obtained dry preparation, when dissolved, exhibited the fermentative properties of the original extract. Soon pepsin was obtained from gastric juice by Schwann (1836), and then a number of other enzymes. During this period, two categories of enzymes had already emerged: soluble (e.g., those contained in saliva, gastric juice, malt extract, etc.) and insoluble, for example yeast mass. With the development of the cell theory, when it was established that yeasts represent single-celled microorganisms, a division of enzymes into organized and unorganized (according to French terminology: enzymes and diastases) arose. The concept of the existence of organized enzymes received its full development in the works of Pasteur. All his attempts to reproduce the fermentation process in the absence of living yeast cells, for example by extracting the active principle from them as the saccharifying enzyme was extracted from malt, ended in failure. Hence, Pasteur concluded that such processes as fermentation are not caused by any specific substances, enzymes, but are the result of the aggregate of life processes of the yeast cell: we are dealing not with the action of chemical agents, but with the function of living, organized matter. Liebig took a diametrically opposite viewpoint, categorically denying the role of living cells in fermentation. Liebig asserted that the decomposition of sugar occurring during fermentation is caused not by the activity of living yeasts, but by the decomposition of dying cells. Liebig pointed out that if a soluble enzyme exists in the yeast cell that decomposes sucrose into a mixture of glucose and fructose, why cannot one assume that it contains an enzyme that decomposes glucose into alcohol and carbon dioxide? What Pasteur and a number of other researchers failed to accomplish was carried out by Buchner, who by high pressure extracted a juice from yeasts that did not contain viable cells and yet rapidly fermented sugar. This established that fermentation is not determined by the entire integral process of cell life activity, as Pasteur taught, but by the action of the enzyme complex contained in the cell—"zymase," which can be separated from the cell and while retaining its activity. The concept that manifestations of cell life activity are the result of the action of a more or less complex aggregate of enzymes, as Liebig believed, became firmly established in science from the moment of Buchner's discovery, and attempts to deny the possibility of cell-free fermentation (Kostychev) were invariably refuted. This removed the opposition between unorganized and organized enzymes, or so-called enzymes (Kuhne). The terms "enzyme" and "enzyme" are at present complete synonyms and are used alongside each other. One can only speak of endo- and exoenzymes: the latter are freely secreted by the cell outward, while the former in normal conditions do not penetrate through the cell membrane but can be released from the other components of the cell after its structure is destroyed. Examples of exoenzymes can be all enzymes of the digestive tract; endoenzymes are the enzymes comprising the zymase complex, endotrypsin of yeasts, etc. Individual enzymes, or even a known portion of the total amount of a particular enzyme, may exhibit varying degrees of connection with the structural elements of the cell. Correspondingly, Willstätter in recent years proposes to distinguish between lyoenzymes, which in the cell are in a free, dissolved or only weakly bound (e.g. adsorbed) state, and desmoenzymes, which are firmly bound to certain components of the cell; these enzymes can be obtained free only after sufficiently deep disintegration of the corresponding components of the cell substance. The nomenclature of individual enzymes is currently built on the principle proposed by Duclaux; all enzyme names are characterized by the ending "ase" added to the root of a word indicating either the substance on which the enzyme acts (amylase for acting on starch—amylum; protease for acting on proteins, etc.), or the process caused by the given enzyme (oxidases—causing oxidation, hydrolases—causing hydrolysis, etc.). In a number of cases, names given before the introduction of the above rational nomenclature have been retained, such as "pepsin," "trypsin," "diastase," "ptyalin," etc. They are retained for historical tradition and sometimes are kept only as a convenient designation for aggregates of enzymes united by physiological moments (e.g. trypsin—a series of proteases contained in pancreatic juice; zymase—the entire complex of fermentation enzymes). The classification of enzymes is built on the same features as the above nomenclature, i.e. a) according to the nature of action and b) according to the nature of substrates, i.e. substances on which the action of the enzyme is directed. According to their action, enzymes are divided into two large groups: hydrolases (see) and desmolases (see). The former cause the hydrolytic cleavage of high-molecular-weight substances (proteins, fats, carbohydrates) into their primary structural elements, breaking the bonds between C and O atoms (in fats and polysaccharides) or between C and N (in proteins and peptides) without violating C-C bonds, i.e. without affecting the actual carbon skeleton of the organic molecule. These are as a rule reactions with negligible energy effect. They predominate in the processes of digestion, in the structural exchange of the cell and play a preparatory role in the energy exchange. Desmolases should be understood, if the term is applied literally, as enzymes causing the actual breaking of carbon bonds, as for example in the decomposition of a hexose molecule into three-carbon compounds or in decarboxylation (see). However, practice has led to the fact that not only these enzymes but also all those that, without causing the breaking of C-C bonds themselves, directly prepare for it or continue it further—in short, the entire complex of enzymes of final decomposition—are included in the group of desmolases. The processes catalyzed by desmolases include all the most important processes of the cell's energy exchange, above all various oxidation-reduction processes. This classification cannot be considered perfect and it cannot always be consistently carried out; for example, the question of which group to assign enzymes that catalyze such hydrolytic processes as proceed with a high energy effect and serve as important sources of energy in the cell (e.g. decomposition of phosphagen, transformations of adenosine pyrophosphoric acid, etc.) is unclear; by biological function these enzymes could be classified as desmolases, all the more so as some of the mentioned transformations themselves enter as links in the chain of desmolytic processes, while by the nature of the reaction they should be classified as hydrolases. The table below gives in schematic form the basic classification of enzymes, their main groups and most important individual representatives. Enzymes A. Hydrolases B. Desmolases I. Esterases I. Zymases Lipases, proper esterases, tan- a) Yeast (alcohol nase, chlorophyllase, etc. fermentation) II. Carbohydrases b) Animal (glycolytic 1. Disaccharides fermentative complex) α- and β-glucosidases Their components: hexokinase, zymohexase, mutases, carbo- α- and β-galactosidases xylase, carboligase α-fructosidase xylose, carboligase Sucrose, maltase, lactase, etc. II. Oxido-reductases 2. Polysaccharides 1. Anoxitropic dehydrogenases (oxi- Amylase, inulinase, cellulose dative-reductive and other slases (polysaccharides) enzymes acting without 3. "

Glucosidases, oxygen-independent: synonyms: hydrolases, reductases, dehydrogenases, dehydrases. 1. Proteases a) Pepsinases (pepsin) b) Trypsases (trypsin) c) Papainases (papain, cathepsin) 2. Peptidases Dipeptidases, polypeptidases 3. Catalase IV. Amidases Urease, histozyme, arginase and others. Detection and quantitative determination of enzymes. With the single exception of most enzymes, no specific characteristics (chemical reactions or physical properties) are known that could serve for their qualitative detection; the only such characteristic is their catalytic activity itself. Only by the presence or absence of the corresponding action can it be concluded whether a given enzyme is present in the material being studied or not. In this case, a negative result cannot be conclusive, since there is always the possibility that an enzyme actually present may, for one reason or another, temporarily be unable to manifest its action. All ordinary methods for quantitative determination of enzymes are also based on measuring their action: the amount of enzyme is judged by the intensity of its action, assuming that there is a direct proportionality between them. This assumption is valid only with very large limitations. In all such measurements, it must be taken into account that what is determined is not the amount, but the activity of the enzyme; the latter can vary within very wide limits, and the same amount of enzyme, depending on a number of external conditions, can give completely different effects. Therefore, it is an essential requirement to equalize, as much as possible, all factors affecting enzyme activity (temperature, active reaction of the medium, concentration of all components; in other cases, 'equalizing activation' is used by adding special activators, etc.). But even under these conditions, only within certain quantitative ratios between the enzyme and substrate does a linear dependence exist between the amount of enzyme and the magnitude of the effect it produces. In other cases, this dependence can acquire a very complex character (cf. Borisov's rule). The amounts of enzyme measured by their action cannot be expressed in absolute units (grams, moles), and one has to resort to conventional units. Since the action of an enzyme consists in accelerating a certain process, and the characteristic of the speed of a chemical transformation is the reaction rate constant, the most perfect method for quantitative determination of an enzyme should be the determination of the rate constant of the reaction it catalyzes. Usually, however, simpler methods are used to judge the speed of the reaction. The criterion taken is either the amount of substrate decomposed in a certain period of time, or, what is preferable (but more complicated), the time required for the transformation of a certain amount of substrate; or finally, the amount of the enzyme material being tested that carries out the transformation of a certain amount of substrate in a certain period of time ('the principle of decreasing series' - see Willgerodt's method or Gross-Fuld's method). The values obtained by this method are taken as the 'enzyme unit,' and the content of the enzyme is expressed in terms of the number of these 'units' per unit of volume or weight (usually of the dry residue) of the enzyme preparation. The chemical nature of enzymes is beginning to be elucidated, and that only for a small number of them, in the very recent years. The main path to studying it lies in isolating the enzyme from natural material and further purification. The criterion of purification again is the measurement of activity: the purer the preparation, the higher the activity should be, relative to dry weight, i.e., the more 'units' should be contained, for example, in 1 g of dry substance of the preparation. Attempts to isolate enzymes by chemical means, for example, by obtaining insoluble compounds of them with certain chemical substances (for this, some dyes, such as safranine, etc., were tried to be used) and then regenerating the active enzyme from the resulting precipitates, have not given satisfactory results. Therefore, and also in view of the extreme lability of enzymes, physical-chemical methods are predominantly used for their isolation and purification, in particular the adsorption method. The adsorption principle was first successfully used by Danilevsky for the separation of pancreatic enzymes. This method reached its highest perfection and development, both practical and theoretical, in the research of Willstätter and his school. The principle of the method is that a solution containing the enzyme in a mixture with all sorts of foreign substances, as is the case, for example, in the natural secretions of glands or in extracts (aqueous, glycerin) from material rich in enzymes, is first subjected to preliminary rough purification; crystalloids are removed by dialysis, the main mass of impurities by precipitation with alcohol, acetone, lead acetate, or some adsorbing substances. Then the enzyme itself is adsorbed, choosing the nature of the adsorbent and the conditions (pH, concentrations, overall composition of the solution) so that selective adsorption takes place as much as possible, i.e., the enzyme is predominantly adsorbed, while impurities remain in the solution. From the resulting adsorption complex (adsorbate), the enzyme is extracted (eluted) with suitable solvents, again striving for selective liberation of the enzyme so that the impurities adsorbed along with it do not pass into the solution. By applying these adsorption and elution processes systematically, varying the adsorbents and conditions, it is possible to free enzymes more and more from foreign impurities. As adsorbents, specially prepared preparations of aluminum hydroxide, kaolin, iron hydroxide, charcoal, etc., have found the greatest application; for elution, solutions of ammonia, phosphates, etc., are used. It is precisely the adsorption method that has made it possible to obtain the most highly purified enzyme preparations known to date, in some cases increasing their degree of purity more than 20,000 times compared to the original material. Despite all its perfection, this method did not lead to the expected goal - obtaining the enzyme in a pure, chemically individual state. The limitation is due to the fact that at higher stages of purification, with the removal of the last remnants of 'accompanying substances' (Begleitstoffe), gradual inactivation of the enzyme begins, and thus the only characteristic by which the presence of the enzyme itself can be detected is lost. Apparently, if some of the accompanying substances are simply foreign, ballast, indifferent material, others have a certain activating or stabilizing effect on the enzyme, and their removal leads to its inactivation. Even the purest preparations obtained by this method, such as Willstätter's peroxidase, undoubtedly consisted for the most part of inert, foreign material, and the content of the enzyme itself in them, according to some calculations, can be estimated at no more than 2-3%. On the other hand, preparative methods gave new ideas about the general physicochemical structure of the enzyme particle. This particle must be considered as a complex consisting of two components. There is an enzyme molecule containing specific, catalytically active chemical groups, adsorbed on a large colloidal 'carrier'; the latter itself is not active, and its role was first seen in that it stabilizes, protects from deactivation the labile active group of the enzyme. Such a complex is something analogous to complex proteins-proteids, with the corresponding enzyme playing the role of the prosthetic group. Research of the most recent times has shown that the colloidal carrier to a large, possibly even decisive, extent determines the magnitude of the catalytic activity of the enzyme's active group and the very character of it. The properties of the enzyme are the properties of the entire particle as a whole, and not only of its so-called active group. The indications that were sometimes encountered that one colloidal carrier in the enzyme molecule could be replaced by another very much need verification and hardly correspond to reality. Probably here the talk was about more random accompanying substances, and not about the carrier itself as an integral part of the enzyme molecule. The active group apparently can be very stable, not decompose spontaneously, and reunite with the carrier into the original complex with unchanged enzymatic activity. Evidence for this was given in brilliant recent works (Theorell) from Warburg's laboratory. A completely different path of preparative obtaining of enzymes has been applied by American researchers Sumner and Northrop. By fractional salting out with ammonium sulfate or precipitation with acetone of concentrated solutions of unpurified enzyme preparations (usually simply commercial ones) - urease, pepsin, trypsin - it is possible to obtain microcrystalline precipitates containing a significant part of the original enzyme. These precipitates are typical protein crystals (see).

They can be recrystallized multiple times without losing (and initially even increasing) their enzymatic activity, from which the authors conclude that the crystals truly represent the enzyme as such. Since no characteristic prosthetic groups can be detected in the composition of the crystalline protein preparation, Northrop assumes that the enzymatic activity is due to a specific combination and arrangement of amino acids in the enzyme protein molecule. The identity of the enzyme with the protein from which the crystals are composed, postulated by Northrop, has not yet found acceptance in enzymological literature and encounters a number of objections. It is quite possible, although also not finally proven, that here there is simply an adsorption of the enzyme on the protein micelles and its involvement together with the latter during crystallization in the composition of the crystals. For elucidating the chemical nature of enzymes, these 'crystalline enzymes' have so far given nothing. The described preparatory methods do not make it possible to approach the elucidation of the chemical nature of the active group of the overwhelming majority of enzymes, because, as indicated, the latter, when attempts are made to separate it from the colloidal carrier, loses its activity and thus escapes further detection; and analysis of the entire complex as a whole cannot give any indications regarding the chemical structure of the actual active group of the enzyme. The path to this study, at least with respect to some enzymes, was opened when independent and stable signs of the active enzyme group, independent of activity, were discovered. Such a sign was the ability of this group or its derivatives to absorb rays of a certain wavelength, i.e., coloration. Warburg established that the respiratory enzyme is inhibited by carbon monoxide, and this inhibition is stronger in the dark than in the light. Carbon monoxide combines with the active group of the enzyme, and this enzymatically inactive compound decomposes in the light. Light can act only on substances that absorb it, and only to the extent of the degree of absorption. By measuring the action of rays with different wavelengths, it became possible to determine which of them are absorbed by the carbon monoxide compound of the enzyme, i.e., the absorption spectrum of this compound was established, albeit indirectly. It turned out to be very close to the absorption spectrum (measured already directly) of the carbon monoxide compound of hemochromogen (the prosthetic group of hemoglobin). This established that the active group of the respiratory enzyme has a structure similar to hemochromogen, hematoporphyrin or hemin. In its structural details, it differs from the pigment part of hemoglobin and remains finally not fully elucidated. Preparatory isolation of the enzyme has not yet been achieved due to its small content and the presence in the cell of large amounts of very similar in properties, difficult to separate, but enzymatically inactive hemin compounds; for example, in yeast, according to Warburg's calculations, the enzyme constitutes only 1/25 of the total amount of hemins contained in the cell. The elucidation of the structural details is possible for now only indirectly—by comparing the absorption spectrum of the enzyme with the spectrum of hemins obtained artificially or isolated from any natural sources; the closest were the spectra of hemins from the blood pigment of marine worms (chlorocruorin) and hemin, synthetically obtained by introducing iron into plant-derived porphyrins (Fig. 1). The hemin nature of the active group was also established for two other oxidation enzymes.

Figure 1. Spectrum of the respiratory enzyme (effect of illumination at equal quantum intensity).

of oxidative metabolism, catalase and peroxidase. Finally, for one of the enzymes of anaerobic metabolism participating in butyric acid fermentation, the presence of an iron atom in its active group has also been established. It has not yet been determined whether it is present here as part of a hemin or in some other grouping (Kubowitz). Directly, by direct observation, Warburg established the spectrum of yet another enzyme widely distributed in cells, the so-called "yellow respiratory enzyme." In this case too, the color was found to belong to the active group of the enzyme; it persists even after the cleavage of this group from the colloidal carrier, which is a protein with globulin properties, whereas the enzymatic activity is lost in the process. Due to the high content of enzymes in certain cells (e.g., in yeast), it could be obtained preparatively in significant quantities. Guided by the color of the active group, it was possible, by cleaving it from the carrier, to isolate it, obtain it in a crystalline, chemically pure form, and subject it to chemical analysis. It turned out to be related to vitamin B₂ (Kuhn and co-workers) and belongs to the group of widely distributed lyochrome pigments or flavins (see), representing a phosphoric acid ester of one of the flavins; its elementary composition is C₁₇H₂₀N₄O₉P. It was precisely with this enzyme that the experiments described above on the cleavage and reunification of its components were carried out. Since a close flavin derivative has recently been synthesized by Kuhn, it can be assumed that it will soon be possible for the first time to obtain an enzyme artificially by synthetically connecting the obtained active group (flavin-phosphoric acid) with the natural carrier protein (Fig. 2). Preparations of crystalline enzymes (see above)—urease, pepsin—also give absorption spectra, but not in the visible, but in the ultraviolet region. These spectra were established both by indirect methods (by measuring the inactivating effect of rays of different wavelengths) and by direct spectrography; both methods give identical results. The obtained spectra are very similar to each other and close to the spectrum of tyrosine; one might think that here, unlike the spectra of hemin and flavin enzymes, the spectrum of the colloidal carrier is determined, rather than that of the active group. Valuable concepts on the structure and chemical nature of enzymes were also provided by the study of so-called "enzyme models." Iron-containing carbon served Warburg as a prototype for the structure of the respiratory enzyme. The action of the enzyme carboxylase (see) turned out to be reproducible using synthetically obtained substituted amines (Langenbeck). Langenbeck suggests distinguishing between active and-activating groups in the enzyme molecule. The former are those chemical groupings that interact with the substrate and are directly responsible for the catalytic effect; the activating groups only enhance the catalytic function of these active groups, much like in dye chemistry the so-called auxochrome groups enhance the coloring power of the chromophore group of the dye itself. By systematic study of the influence of substituting, "activating" groups in such models on the catalytic ability of the latter, it was possible to increase it to a very high degree, up to 4,000 times compared to lower, unsubstituted amines. Calculated per mole of converted substrate per minute, such models turn out to be only 20 times weaker than, for example, Northrop's crystalline enzymes, which, given the already achieved thousandfold increase in activity, appears to be a difference that will be easily overcome. It seems plausible to assume that natural carboxylase also has a similar structure, that the active "zymophoric" group that directly determines the catalytic action is also the amine group here, but that the activating groups are incomparably more effective than those found to date in synthetic experiments. This concept outlines yet another mechanism for the emergence of the unusually high catalytic power of the enzyme: the influence not only of the colloidal carrier, but also of auxiliary activating groups that are part of the enzymatically active part of the molecule itself. Regarding the chemical nature of the active group of other enzymes, apart from those specified above, no definite concepts yet exist. Attempts to approach its elucidation by studying the effect of various compounds serving as reagents for specific chemical groupings (reagents for the amino group, for aldehyde, acid, basic groups) have not yet yielded sufficiently clear and convincing results. The magnitude of the molecular weight of enzymes is practically determined by the dimensions of their colloidal carrier, which constitutes the bulk of the enzyme molecule. Both chemical and physical methods are used to measure molecular weight. The first are based on the action of enzyme-inactivating compounds, e.g., salts of certain metals, and the calculation of "equivalent weight"; with the discovery of the hemin nature of certain enzymes, it became possible to calculate the molecular weight from the iron content. Of the physical methods, diffusion rate measurements were used, and in recent times, the sedimentation method using Svedberg's ultracentrifuge. The obtained values lie within the limits usually encountered for proteins. Some data are compared in the accompanying table. Enzyme / Measurement method / Molecular weight / Author / Sucrase: Silver inactivation, 5,000, Euler; Diffusion rate, 20,000, Euler; Diffusion rate, 50,000, Melvyn-Hughes. Pepsin crystalline: Osmotic pressure, Diffusion rate, Content of Cl and S, 36,000, Northrop. Catalase: Diffusion rate, 69,000, Stern. Urease: Silver inactivation, >40,000, Semper and Mürbach. Yellow respiratory enzyme: Sedimentation in Warburg's ultracentrifuge, 50,000 and 70,000, Svedberg. Properties of enzymes. Enzymes as catalysts. In enzymes, the fundamental property of catalysts is expressed to an especially prominent degree: the ability in negligible quantities to cause a huge effect in terms of accelerating a reaction. Already old observations, referring to very crude preparations, indicated that, for example, one part of rennet (chymosin) is capable of causing the coagulation of 400,000 parts of milk; the action of the "85,639th enzyme" isolated by Meldrum and Roughton, which catalyzes the evolution of CO₂ from H₂CO₃ solutions, is detected even at a dilution of 1 : 10,000,000; the purest preparations of peroxidase and catalase decompose in 1 sec. more than a 200-fold weight amount of hydrogen peroxide. These calculations still refer to preparations that undoubtedly contain a very significant amount of foreign substances and in which the content of the enzyme itself hardly exceeds a fraction of a percent. Recalculations to actual gram-moles of the enzyme (which in some cases can be done with sufficient approximation) show even significantly higher values: thus, in 1 sec. one mole of sucrase at 40° cleaves 1,000 moles of cane sugar; for a number of enzymes participating in oxidative processes (Warburg's respiratory enzyme, catalase, peroxidase, the aforementioned Meldrum-Roughton enzyme), values on the order of 10,000-100,000 moles of substrate transformed by one mole of enzyme per second are obtained. This almost achieves the generally possible limit of catalytic efficiency, since we are dealing here with an order of magnitude corresponding to the total number of collisions of enzyme molecules with the substrate. Thermolability is one of the most characteristic properties of enzymes. It is impossible to indicate any definite inactivation temperature of a particular enzyme below which inactivation would not proceed and upon reaching which it would instantly occur. Inactivation is a function of both the temperature and the duration of its action, differing, however, like the protein denaturation reaction, by an extraordinarily high temperature coefficient; thus, for example, for milk peroxidase it reaches (measured at 70°) 3,040 compared to values of 2-3 characteristic of ordinary chemical reactions. Therefore, upon reaching a certain temperature, different for different enzymes, inactivation proceeds with extreme rapidity. If we take as a criterion for comparison the "critical temperature"—the one at which the enzyme loses half its activity in an hour—we obtain the following values for a number of enzymes: lipase (dry) - 151°, emulsion dry - 101°, emulsion wet - 54°, peroxidase - 69°, trypsin - 65°, pepsin - 65°, sucrase - 59°. For comparison, the critical denaturation temperatures of two proteins can be cited: egg albumin - 76°, hemoglobin - 63°. In a dry state, enzymes withstand, as can be seen from the given figures, heating above 100°; in solutions at this temperature, almost instantaneous inactivation occurs (with rare exceptions). The presence of the substrate often increases the heat resistance of the enzyme; the rate of thermal inactivation also depends to a very strong degree on the active reaction (pH) of the medium.

The similarity in the temperature dependence of enzyme inactivation and protein denaturation suggests that the action of temperature is directed not so much at the active group of the enzyme as at the colloidal carrier and associated substances. This is confirmed by the fact that temperature sensitivity varies with the degree of enzyme purification: in some cases, it increases upon purification (here one might think that associated substances acting as protective colloids and preventing changes in the colloidal carrier of the enzyme are removed), while in other cases, conversely, the purified enzyme proves to be more resistant to an increase in temperature; it is possible that in unpurified preparations, mechanical entrapment of the enzyme by coagulating associated substances occurs. Thermolability of enzymes also accounts for the phenomenon of the temperature optimum, which was considered characteristic of enzymes for a long time. Like any ordinary chemical reaction, reactions catalyzed by enzymes increase in rate with an increase in temperature. The temperature coefficient of enzymatic reactions generally lies somewhat lower than that of the same reactions proceeding without the participation of enzymes; nevertheless, it lies within the same limits that are usual for chemical processes, Q₁₀ = 1.5 to 3. Along with this acceleration of the reaction itself, as temperature increases, the rate of enzyme inactivation also increases, possessing, as indicated above, an extremely large temperature coefficient. Upon reaching a certain temperature, the second factor first balances and then overtakes the first, and a deceleration followed by complete cessation of the process begins. The position of the observed temperature optimum can vary greatly depending on the duration of observation: the shorter it is, the less thermal inactivation of the enzyme affects it, and the higher the temperature at which the found optimum lies, and vice versa. Thermal inactivation is generally irreversible. The described cases of gradual reactivation after the cessation of high temperature action are unclear in mechanism and allow for various interpretations. Enzyme activity depends extremely strongly on the concentration of hydrogen ions (pH). With a change in pH, enzyme activity changes regularly, reaching a maximum at a certain pH value. The position of this optimum varies for different enzymes, for the most part lying within the pH range between 5 and 7. Below are the optimum pH values for some of the most important enzymes: Enzyme Enzyme Amylase (saliva) 1.5-3.6 Amylase (blood) 8.0-11.0 Amylase (malt) 3.0-6.0 Lipase (gastric juice) 6.0 Same, purified 6.2 Lipase (pancreatic) 4.6-5.0 Phosphatases (animal) 6.1 Phosphatases (Aspergillus) 6.6 Arginase 4.6-5.0 Carboxylase 4.1-4.5 Trypsin 4.5-6.5 Cathepsin 6.9-7.0 Sucrase (yeast) 6.8 Maltase (yeast) 5.2 Maltase (malt) 6.0 beta-Glucosidase 8.0 Trypsin 7.0-8.6 Cathepsin 7.2-9.4 Sucrase (yeast) 3.0-5.5 Maltase (yeast) 7.2-7.5 Maltase (malt) 9.8 Urease 4.8 The course of curves depicting enzyme activity as a function of pH in many cases reproduces the character of dissociation curves of an amphoteric electrolyte depending on the concentration of hydrogen ions. This served as the basis for the theory developed by Michaelis, according to which enzymes are regarded as ampholytes, with only undissociated particles (or those possessing only a small positive or negative charge) normally being active; the optimum action of the latter must consequently coincide with or lie close to the isoelectric point (see). This view is supported by observations of the cataphoretic transfer of enzymes: within the optimal pH zone, enzyme particles remain stationary in an electric field. However, this latter regularity is not always observed, since the cataphoretic charge of an enzyme can be determined not only by the dissociation of its active group, but also by the state and character of more or less closely associated accompanying substances. With the removal of the latter during enzyme purification, its cataphoretic properties can change more strongly than the dependence of activity on pH, although the latter also does not always remain unchanged (cf., for example, the data given above for gastric juice lipase, where the pH optimum shifts by a full two units upon purification). It must be admitted that sometimes the influence of pH is manifested through an effect on the substrate, specifically in cases where the latter possesses electrolyte properties, such as proteins and peptides. According to Northrop, the dependence of proteinase action (pepsin, trypsin, cathepsin) on pH does not boil down to a change in the state of the enzyme itself, but is due to the varying degree of ionization of the substrate; accordingly, the optimum action of the same enzyme may turn out to lie at different pH values with different substrates, depending on the electrical properties of the cleaved protein (cf. Proteases). The extremely strong influence of hydrogen ion concentration on enzyme activity makes it necessary in all work with enzymes to pay special attention to maintaining a defined pH, which is achieved by adding appropriate buffer solutions. Numerous earlier studies conducted without observing this condition prove to be largely valueless. A factor whose significance for enzyme activity was only recently recognized is further the oxidation-reduction potential of the medium in which the enzyme action takes place. This is manifested in the influence that the addition of substances (or systems) possessing oxidizing or reducing action exerts on a number of enzymes. It can be assumed that enzyme activity is determined not only by the degree of its electrolytic dissociation, but also by whether the enzyme is in an oxidized or reduced state. The available observations, summarized in the following table, are so far only qualitative in character, and much still needs to be clarified and refined. Further development of research in this direction will undoubtedly be able to significantly broaden our concepts of the nature of the active group of enzymes and their mechanism of action. There are indications, however, that in some cases the action of oxidation-reduction agents is directed not at the enzyme itself, but at various impurities, such as traces of heavy metals. Data on the action of radiant energy on enzymes are contradictory and indefinite (with the exception of the studies described above on the action of light on compounds of the respiratory enzyme with carbon monoxide). They do not provide an opportunity to draw any generalizing conclusions. Usually, more or less significant inactivation is observed, and more rarely, some increase in activity. Visible light has a weak effect (it can be enhanced in the presence of sensitizers such as eosin, rose bengal, etc.); ultraviolet rays act more strongly. Enzyme Inhibitors Activators Papain Cathepsin Amylase Ascorbic acid Glutathione (G-S-S-G) (?) Phosphatase Sulfhydryl compounds (-SH) Methylglyoxalase Glutathione (GSH) Arginase Oxygen; HCN, cysteine, glutathione (GSH) at pH below 7 Glutathione + metals (Fe, Cu); cysteine + metals; ascorbic acid + Cu Cysteine at pH 9.4 Urease Methylene blue, oxidation products of polyphenols (quinones); hydrogen peroxide Catalase Ascorbic acid; sulfhydryl compounds Zymase, glycolytic enzyme Iodine, dyes with high oxidation-reduction potential Sulfhydryl compounds (?) In the latter case, a regular dependence of inactivation on wavelength is sometimes observed, and it is thus possible to obtain a definite "inactivation spectrum" of the enzyme. The spectrum found by this way for a urease preparation turned out to be practically identical with the absorption spectrum obtained by direct measurement of absorption (Kubowitz, Haas). For mitogenetic rays (see), an inhibiting effect is detected in the majority of cases (Mardashev). On the other hand, the enzymatic processes themselves are, according to the work of Gurwitsch's laboratory, sources of mitogenetic radiation, with each enzyme possessing its own specific emission spectrum (Fig. 3). Figure 3. Mitogenetic emission spectra during enzyme action. Surface-active agents often noticeably inhibit enzyme action, apparently displacing the substrate from the active surfaces of the enzyme micelle. This was demonstrated particularly clearly in Warburg's experiments, where the inhibiting effect of narcotics on respiration was found to be in direct proportion to the size of the area occupied by the adsorbed capillary-active substance in the surface layer. Adsorption phenomena in general must be ascribed the significance of an extremely important factor influencing the intensity of enzymatic processes. A definite spatial orientation of the substrate in the adsorption layer and the associated changes in its molecular structure create the necessary prerequisite for enzyme action (cf. below). It must be assumed that usually this orientation of substrate molecules is carried out on the surface of the enzyme micelle.

As the remarkable experiments of Schulman and Rideal on proteinases have shown, upon the destruction of the colloidal part of the micelle by digestion or thermal denaturation, the active group of the enzyme loses its ability to act on dissolved protein. But such enzymes, inactivated in the usual sense, continue with almost unchanged activity to digest protein when its molecules are in the form of a spatially oriented layer at the medium-air interface. If adsorption phenomena influence the substrate here, then under the natural conditions of enzyme action in the living cell, a powerful factor regulating enzymatic activity is the adsorption bond of the enzyme itself with the structural material of the cell, with the components of its protoplasm (Oparin). The specificity of enzyme action, i.e., the ability of each of them to catalyze only a strictly defined chemical reaction, belongs to the number of the most characteristic properties of enzymes, although to one degree or another it can also be found in simpler catalysts. This specificity can manifest itself in various ways. The most frequent case is that on a given substrate (we may be talking about some single substance or a group of compounds related in chemical structure) only one specific enzyme "set" for it acts, which does not act on other substances. Such relationships between the enzyme and the substrate served to compare them with the relationship between a lock and a key (E. Fischer). This kind of specificity, determined by the structure of the substrate, can achieve varying degrees of selectivity. At one time, the tendency dominated (and still often manifests itself) to postulate for each individual substance undergoing biochemical transformation the existence of a specific enzyme directing its action only to the given compound. In some cases, such a limiting selectivity of action undoubtedly exists; thus, for example, urease acts exclusively on urea, catalase only on hydrogen peroxide, and so on. Selectivity reaches its highest degree when the enzyme acts on only one of two stereoisomers of the same substance. However, much more often we deal with the specific orientation of the enzyme not to any definite compound, but to a known chemical grouping within a complex molecule, to a certain chemical function; such functions can occur in the composition of various compounds and then the enzyme will act on all these substances. We have such a character of specificity—orientation to a certain chemical grouping in the substrate molecule—for example, in proteases and peptidases (see Proteases), where the site of enzyme action is the -C-N- bond, in esterases acting on the R-C-O-R bond, and so on. On the basis of this, it appears possible in certain cases to significantly reduce the number of supposedly separate enzymes and correspondingly simplify the classification. Thus, for example, in the carbohydrate group, the existence of separate enzymes was assumed for each individual complex carbohydrate. According to Weidenhagen, however, there is a much more limited number of enzymes here, the specificity of action of which is determined by the following features of carbohydrate structure: the nature of the glycosidic hexose, the arrangement of the oxygen bridge in it, and the configuration of the glycosidic bond (alpha- and beta-isomerism); therefore, there is only one enzyme, alpha-glucosidase, which splits both maltose and alpha-glucosides, and even sucrose, since the latter contains an alpha-glucosidic grouping in its diglycosidic bond (cf. Disaccharides). In other cases, the specificity of enzymes is manifested not in the nature of the substrates to which their action is directed, but in the nature of the transformations of these substrates caused by various enzymes: the same substrate can be subjected to the action of different enzymes, each of which causes its own specific transformation of the substrate. Thus, for example, if the trisaccharide raffinose, having the structure: galactose-glucose-fructose (melibiose-sucrose), is acted upon by the enzyme sucrase, the molecule disintegrates at point II with the formation of melibiose and fructose; upon the action of the enzyme melibiase, the cleavage goes at point I with the formation of galactose and sucrose; in the same way, pancreatic amylase splits starch with the formation of alpha-maltose, and malt amylase gives beta-maltose; from grape sugar under the influence of yeast zymase, alcohol and CO₂ are obtained, and under the influence of animal zymase, lactic acid. The basis of specificity, especially in cases where fine differences in substrate structure are concerned (e.g., stereochemical), should undoubtedly be sought in the definite configuration of the active group of the enzyme. Along with this, the aforementioned "activating" groups may also play a certain role. In some cases, the sphere of activity of an enzyme, i.e., the limit of its specificity, can change depending on the presence or absence of auxiliary substances—activators, kinases, coenzymes; however, usually the action of these agents is mainly expressed in changes in the magnitude of enzyme activity, and the same applies to the so-called antiferments (see).

Reversibility of enzyme action. The reactions catalyzed by enzymes mostly belong to the class of reversible reactions. Since a catalyst does not introduce measurable amounts of energy into the reaction system, it cannot shift the equilibrium position of a reversible reaction and therefore must accelerate both its phases—both the forward and the reverse reaction. This rule remains valid for biological catalysts—enzymes. Consequently, if we start from concentrations of cleavage products exceeding the equilibrium concentrations, the enzyme must also catalyze the synthetic process. This is not always observed, since in many enzymatic reactions the equilibrium position is shifted almost entirely towards cleavage. In those cases where the equilibrium position lies within measurable limits, the synthetic action of the enzyme is easily detected, as for example in the action of lipases, glycosidases, zymohexase, and others. Exhibiting stereochemical specificity with respect to cleaved substrates, enzymes also manifest it in synthesis, utilizing from the racemic mixture predominantly only one or another isomer and thus carrying out asymmetric synthesis (see). Along with the described simplest mechanism of synthesis by catalyzing a reversible reaction, more complex syntheses are also carried out with the participation of enzymes, proceeding by the type of coupled reactions. Examples of these can serve the synthesis of carbohydrate from lactic acid in muscle (Pasteur-Meyerhof reaction), the resynthesis of other active substances in muscle (phosphagen, adenylyl pyrophosphate—Lohmann reaction), the synthesis of amino acids from nitrogen-free products, and so on.

Theory of enzyme action. The interpretation of the mechanism of action and the peculiar features of enzymatic catalysis must properly take into account its most characteristic features, which are: 1) the extremely high efficiency of enzymes as catalysts, manifested in the huge absolute velocities of the reactions catalyzed by them, 2) the limiting specificity of action both in the sense of a strictly limited choice of substrate and in the sense of an equally strictly maintained direction of the reaction, proceeding unlike what is often observed in ordinary catalysis, with negligible formation of any "side" products. At the same time, since enzymatic action is a peculiar, yet only a particular case of catalysis in general, the interpretation of its nature must necessarily be based on general ideas about the essence of catalysis (see). At the present time, one can speak of three main theories of enzyme action: the intermediate compound theory, the adsorption theory, and theories based on molecular-kinetic concepts. None of them can be recognized as exhaustive; they complement each other, but do not yet provide the opportunity to construct a unified general theory of enzymatic action. The classical intermediate compound theory, which generally dates back in the general theory of catalysis to the first research in this direction, is also the basic and most accepted theory of enzymatic catalysis at present. Its fullest development as applied to enzymatic catalysis was obtained in the works of Michaelis. Michaelis assumes the formation of an intermediate compound of the enzyme with the substrate S: F + S ⇌ FS. (1) The second stage, constituting the very essence of the enzymatic reaction, is the decomposition of this complex with the formation of the product (P) of substrate transformation and with the regeneration of the free enzyme ready for further action: FS → F + P.

(2) W. M. E. Vol. XXXIII. Michaelis's theory proved to be very fruitful. It made it possible to interpret the conditions for the reversibility of enzyme action, the inhibition of enzyme action by the products of enzymatic reactions or by substances similar to them, which also have an affinity for the enzyme (so-called competitive or inhibitory competition), the action of poisons on enzymes, etc. At present, it is the most developed and experimentally substantiated theory of enzymatic action. However, as indicated above, it cannot be considered exhaustive. First of all, it must be noted that the basic equations of this theory formally coincide with the expressions (equations of the Langmuir adsorption isotherm), based on the concept of the adsorptive, rather than chemical, nature of the combination of the enzyme with the substrate. This can serve as an argument in favor of the adsorption theory of enzyme action, which considers this action on the basis of the doctrine of heterogeneous catalysis. Taking into account the above-mentioned significance of the presence of a colloidal carrier for enzyme action, it must be admitted that this action cannot be considered exclusively from the point of view of homogeneous catalysis, as the Michaelis theory does; here we have microheterogeneous catalysis, combining in itself the features of both homogeneous, purely chemical, and macroheterogeneous catalysis. As for the latter, modern concepts have moved far from the initial views, which attributed the cause of the acceleration of the reaction only to the increase in the concentration of reacting substances in the surface layer (Bayliss). In the mechanism of heterogeneous catalysis, we also have moments of a chemical nature. Under the influence of forces acting on the surface of the catalyst, originating from certain chemical groupings, or under the action of electric fields arising here, changes in atomic distances occur in the substrate molecule, certain shifts of valence bonds arise, the latter are weakened, and this prepares the breakdown of the molecule or its interaction with a partner in the chemical reaction. The theory of intermediate compounds well explains the high specificity of enzymes, the adsorption theory properly takes into account the significance of their colloidal nature. But the above exposition of these theories does not yet answer the question about the absolute rates of enzymatic reactions, which, as indicated, reach unusually high values. Recently, a theory of enzyme action by the type of chain reactions has been proposed. According to the theory of Willstätter and Haber, formulated to explain the action of oxidoreductase enzymes, the enzyme forms a free radical from the substrate through monovalent oxidation, which causes the chain reaction to occur; the enzyme itself temporarily passes into a mono-deoxy-form, from which it is then regenerated to its original state. As an example, we can cite the scheme of catalase action: H₂O₂+catalase=HO (first radical)+deoxycatalase; HO+H₂O₂=H₂O+OH (second radical); OH+H₂O₂=H₂O+HO (first radical) and so on. Thus, the enzyme participates only at the first moment-in the formation of the first radical, and then the reaction proceeds already without the participation of the enzyme, until the chain breaks. Similar views were developed by Medvedev in relation to hydrolytic enzymes, however, without specifying the chemical nature of the hypothetical carriers of the chain reaction-"active molecules". The theory of chain reactions in its application to enzyme action well explains the already very high absolute speed of enzymatic transformations, but it encounters a number of serious difficulties, in particular in regard to the interpretation of the specificity of enzyme action; against it, a number of weighty objections have already been raised (A. Bach), and it is now difficult to say to what extent it will prove productive for solving the problem of enzyme action. The biological significance of enzymes is directly clear from their function as the main catalysts in living matter. All manifestations of vital activity, all functions of the living cell and tissue are ultimately based on certain chemical reactions occurring in living matter. The regulation of physiological functions is to a large extent the result of the regulation of the rates of the underlying reactions, and often for the very occurrence of a certain physiological phenomenon, it is required that the speed of the chemical reaction causing it reaches a certain value. It would be incorrect to assert that enzymes are the only catalytically acting agents in the cell and in the organism. A number of agents of a simpler nature are known, lacking one or another of the properties characteristic of enzymes (not colloidal, not thermolabile, little specific, etc.), which also play the role of catalysts in metabolism; but for these simple catalysts, it is characteristic that as a rule they do not manifest their action independently, but participate as auxiliary factors in processes that are ultimately catalyzed again by enzymes. Examples of this can be: glutathione (see), playing the role of an intermediate catalyst in the transfer of hydrogen activated by corresponding enzymes-dehydrogenases; cytochrome, also playing a role in oxidation processes with the participation of enzymes that activate oxygen-oxidases; adenylic acid, catalytically accelerating the breakdown of phosphagen, which again proceeds under the influence of a special enzyme, etc.-As our knowledge expands, new forms of enzyme action are constantly being discovered. Even those that were previously considered purely physical processes, such as gas exchange in the lungs (release of CO₂ from H₂CO₃), turn out to proceed under the influence of a special enzyme. Therefore, it will not be an exaggeration to say that enzymatic action underlies all phases of the life process.

W. Esh'el'gardt.