Colloidal solution of water in organic solvents: a microheterogeneous medium for enzymatic reactions.

To simulate in vitro the conditions under which enzymes act in vivo, enzyme molecules have been entrapped in hydrated reverse micelles of a surfactant in organic solvents. In this system the catalytic activity of one of the enzymes studied (peroxidase) became much higher than in water, and the specificity of the other enzyme (alcohol dehydrogenase) was dramatically altered.

Reactivation of enzymes irreversibly denatured at elevated temperature. Trypsin and alpha-chymotrypsin covalently immobilized on Sepharose 4B and in polyacrylamide gel.

Trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.2) covalently immobilized on Sepharose or in polyacrylamide gel has been irreversibly denatured at 70--90 degrees C and then reactivated in an almost 100% yield. Thermoinactivated enzyme is first made to unfold under the action of urea with S-S bonds being simultaneously reduced and then made to refold (under the optimal conditions for the thiol-disulfide exchange) into its native conformation. It is demonstrated that the 'irreversible monomolecular thermoinactivation-reactivation' cycle can be repeated many times. The contribution of various mechanisms to thermoinactivation of the enzymes is discussed. Based on the data obtained, the irreversible thermoinactivation of enzymes under investigation should be ascribed only to changes in their secondary and teritary structures; the primary structure is not likely to be affected.

Enzymatic synthesis in biphasic aqueous-organic systems. I. Chemical equilibrium shift.

A theoretical analysis of the causes of chemical equilibrium shift (i.e., change in product yield), accompanying replacement of water as the reaction medium by biphasic system "water-water-immiscible organic solvent", is given. A model is described, which is based on equilibrium partition of reagents between the two phases and establishment of a chemical equilibrium. In terms of this model, the apparent equilibrium constant, Kbiphasic which is evolved from the equilibrium concentrations of the reagents referred to the total volume of the biphasic system, should depend on the ratio of the volume of the organic and aqueous phases and the partition coefficients. The theoretical dependences were verified experimentally. Firstly, it was shown that for oxidation of isobutanol into isobutyraldehyde, catalyzed by alcohol dehydrogenase, the equilibrium shift that takes place in a water-hexane biphasic system is determined by the partition coefficients of the reagents found in an independent experiment. Varying the composition of the organic phase (hexane or ethyl acetate and their mixtures), the equilibrium could be shifted (compared to the aqueous solutions) towards both the initial reagents and the end-products: thereby changing the apparent equilibrium constant by two orders of magnitude. Secondly, alpha-chymotrypsin-catalyzed synthesis of ethyl ester of N-benzoyl-L-phenylalanine (from the respective acid and alcohol) in a biphasic system containing chloroform, benzene, carbon tetrachloride or diethyl ether, has been studied. The apparent equilibrium constant has been experimentally demonstrated to depend on the ratio of the aqueous and organic phase volumes in an extreme fashion, as has been predicted by the theory. The yield of the product in the reaction optimum amounts to 100%, whereas in water it is as low as 0.01%.

Chemical modification of the epsilon-amino groups of lysine residues in horseradish peroxidase and its effect on the catalytic properties and thermostability of the enzyme.

Chemical modification of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) (isoenzyme C) by anhydrides of mono- and dicarboxylic acids and picryl sulfonic acid has been performed. The effect of the modification on the catalytic activity, absorption and circular dichroism spectra of peroxidase has been studied. Rate constants of irreversible thermoinactivation (kin) for the native and modified peroxidase at 56--80 degrees C have been measured. The effective values of the thermodynamic activation parameters of thermoinactivation, delta H not equal to and delta S not equal to, have been also determined. A relationship between the number of modified epsilon-amino groups of lysine residues and the nature of the modifier on the one hand, and the conformation and thermostability of the enzyme on the other, is discussed. It has been shown that it is the degree of modification, rather than the nature of the modifier, that produces the major effect on the macromolecular conformation and the thermostability of the enzyme after modification. The conclusion is drawn that the thermostability of the modified enzyme increases due to the decrease of the conformational mobility in the protein moiety around the heme.

Substrate specificity of penicillin amidase from E. coli.

1. The kinetic parameters of 12 substrates of penicillin amidase (penicillin amidohydrolase, EC 3.5.1.11) from E. coli have been determined. Most of the penicillin amidase amide substrates containing a phenylacetyl group in the acyl moiety have been shown to have similar catalytic constants of 50 s-1. Substitution of the phenylacetyl group b 2-thienylacetyl group (cephalothin, cephaloridine) having a similar structure leads to a slight decrease in kcat. 2. Nonspecific penicillin amidase substrates, which contain a free amino group in their acyl moiety, are characterized by a strong dependence of kcat, on the structure of the leaving group with Km being constant. To investigate the free amino group influence on the reaction kinetics, pH-dependences of kcat/Km of enzymatic hydrolysis of phenylacetic and D-(-)-alpha-aminophenylacetic acid p-nitroanilides have been studied. It has been shown that enzyme binds the deprotonated form of the substrate only. 3. Under thermodynamically favourable conditions for the synthesis of beta-lactam antibiotics (at low pH), a concentration of the deprotonated substrate form is very low, and the reaction proceeds in the bimolecular regime. The value of the second-order rate constant for the substrate having a free amino group is small even at pH 7.5, and sharply decreases as does the pH. Hence, despite the favourable thermodynamic conditions for the production of all beta-lactam antibiotics, low reaction rate is the basic hindrance for enzymatic synthesis of penicillins and cephalosporins having a free amino group in the acyl moiety.

Study of the role of arginine residues in bacterial formate dehydrogenase.

Modification of 12 arginine residues per molecule of formate dehydrogenase (formate : NAD+ oxidoreductase, EC 1.2.1.2.) from the methylotrophic bacterium, Achromobacter parvulus I, by 2,3-butanedione results in complete inactivation of the enzyme. Inactivation of the enzyme is reversible and proceeds in two steps via formation of the intermediate enzyme-butanedione complex. Coenzymes but not formate effectively protect formate dehydrogenase from inactivation. Complete maintenance of enzyme activity and specific protection of one arginine residue per enzyme subunit are achieved on formation of the binary complex, enzyme-NAD, or the ternary complex, enzyme-NAD-azide. One arginine residue is supposed to be located at the NAD-binding site of the formate dehydrogenase active centre.

The protoporphyrin-apoperoxidase complex as a horseradish peroxidase analog. A fluorimetric study of the heme pocket.

Similarity of the protein tertiary structures of the native horseradish peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) and protoporphyrin-apoperoxidase complex has been shown on the basis of identity of the tryptophan fluorescence parameter at pH 2.0-8.0 and of the circular dichroism spectra of the two proteins. Absorption and fluorescence spectra have been obtained for protoporphyrin in the complex in the pH range 7.0-1.6. A shift in the apparent pK by 4 units has been observed for protonation of the protoporphyrin pyrrolic ring in the complex. From this shift, the dielectric constant has been evaluated for the heme pocket of the peroxidase (approx. 20). Fluorescence quantum yield of protoporphyrin in the complex increased with pH decreasing from 5.0 to 3.5, whereas the spectrum pattern and fluorescence lifetime did not change. The ions, I- and [Fe(CN)6]-4, peroxidase substrates, did not quench the protoporphyrin fluorescence in the complex at about neutral pH, whereas the quenching markedly enhanced with lowering pH. The bimolecular constant for the I- -quenching of the porphyrin fluorescence on the complex showed a pH-dependence similar to that of the bimolecular rate constant for the reaction of peroxidase compound I with I-. Mechanism for I- oxidation at an acid pH in the presence of peroxidase has been proposed.

Enzymatic mechanochemistry: a new approach to studying the mechanism of enzyme action.

1. Covalent binding of model enzymes, chymotrypsin and trypsin, to elastic polymer supports, nylon and viscose (cellulose) fibers, human hair, methacrylate rubber, has been effectuated. On mechanical stretching of the fibers, the catalytic activity of the enzymes bound to them decreases, and when they relax, it increases to the initial level. The data obtained by us fit the concept that the effect is due to reversible deformation of the bound enzyme molecules induced by fiber stretching. 2. Analysis of the dependence of the catalytic activity of the enzymes chemically bound to the fiber on the degree of fiber deformation shows that the reversible inactivation of the enzymes induced by support stretching occurs even if the deformation of the enzymes' molecules is as small as 0.5 A. 3. The deformation of the enzyme molecules induced by fiber stretching entails a change in the substrate specificity of the biocatalysts, i.e. the activity towards "good" substrates decreases, and towards "poor" substrates increases. 4. The deformation of the enzyme molecules induced by fiber stretching results in a decrease of the specific catalytic activity of the biocatalyst, whereas its thermal stability increases. 5. The results obtained allowed a new, mechanochemical, approach to be suggested for studying major problems of enzymatic catalysis.

Kinetics of the inactivation of the protein-lipid complex, firefly luciferase, by sodium deoxycholate and its reactivation by phosphatidylcholine.

Firefly luciferase has been shown to be a protein-lipid complex. Phospholipids and neutral lipids bound to luciferase have been identified. Sodium deoxycholate rapidly inactivated the enzyme, but an excess of phosphatidylcholine recovered luciferase activity. From the kinetics of inactivation and reactivation, a mechanism for interaction of the enzyme with detergents and phospholipids has been proposed. The substrates ATP and Mg2+ stabilized luciferase during delipidation.

Principles of enzyme stabilization. V. The possibility of enzyme selfstabilization under the action of potentially reversible intramolecular cross-linkages of different length.

The denaturing action of guanidine . HCl on modified alpha-chymotrypsin (EC 3.4.21.1) preparations has been studied. The consecutive treatment of alpha-chymotrypsin with N-acetyl-homocysteine thiolactone, 5,5'-dithio-bis-(2-nitrobenzoic acid) and dithiols of HS-(CH2)n-SH type, with n ranging from 4 to 10, leads to enzyme stabilization as a result of protein modification. A greater stabilization effect can be achieved by enriching the protein molecule with groups reactive towards dithiols, after first modifying carboxygroups. In this case dithiol with n=5 forms an intramolecular cross-linkage. If an equimolecular mixture of different dithiols is used for enzyme modification, the enzyme gradually 'selects' 1,5-dithiol for the formation of an intramolecular cross-linkage instead of the initial one-point modification. The use of potentially reversible cross-linkages may be generally employed for the preparation of stabilized water-soluble enzymes via the mechanism of selfstabilization.

The principles of enzyme stabilization. IV. Modification of 'key' functional groups in the tertiary structure of proteins.

The dependence of alpha-chymotrypsin thermostability and catalytic activity on the degree of its amino groups modification has been studied. Modification was carried out by both alkylation (using acrolein with further reduction of Schiff bases by sodium borohydride) and acylation (with siccinic or acetic anhydrides). It has been determined that modification of the majority of titrated amino groups (approximately 80%) only has a slight effect on the first-order rate-constant characterizing the monomolecular process of enzyme thermoinactivation (50 degrees C, pH 8). Thermostability sharply increases (by 120 times) only for a degree of modification higher than 80%, but, nevertheless, the complete substitution of all the titrated amino groups again leads to enzyme destabilization. The conclusion has been drawn that there is only one or two amino groups out out approximately fifteen titrated ones, the modification of which plays a key role in the lateration by the enzyme of its thermostability. The degree of the stabilization effect has been studied relative to both the nature and concentration of the salt added Na2SO4, NaCl, KCl, CCl3COOK, (CH3)4NBr. Ultraviolet absorption (280 nm) of chymotrypsin has also been elucidated with respect to the degree of alkylation of its NH2-groups. The data obtained allowed the conclusion to be drawn that enzyme modification leads to a decrease in the non-electrostatic (hydrophobic) interactions on the surface layer of the globule. As a result, a protein conformation more stable in respect to denaturation (unfolding), is formed.

The effect of mechanical stretching of the myosin rod component (fragment LMMMM S-2) on the ATPase activity of myosin.

The binding of myosin to nylon fiber gives immobilized myosin with a considerable ATPase activity. Treatment of immobilized enzyme with papain results in the entire ATPase activity (known to be concentrated in myosin heads, (fragment HMM S-1)) being replaced from the fiber into the solution; this means that myosin is chemically bound to the fiber via its rod part (fragment LMM+HMM S-2). When nylon fiber is mechanically stretched, the ATPase activity of myosin attached to it sharply decreases; after relaxation of the fiber the enzymatic activity returns to the initial level. The detailed study of this phenomenon has shown that reversible inactivation of myosin upon fiber stretching is not the result of an altered microenvironment of the enzyme. The discovered regulatory effect is ascribed to deformation of myosin molecules induced by support stretching. Thus deformation of the myosin tail (not indispensable for ATPase since its cleaving-off does not alter the enzymatic activity) leads to decrease in the ATPase activity of the enzyme. The possible role of the above phenomenon in the mechanism of muscle contraction is discussed.

The principles of enzyme stabilization. VI. Catalysis by water-soluble enzymes entrapped into reversed micelles of surfactants in organic solvents.

1. The possibility of stabilizing water-soluble enzymes against the inactivation action of organic solvents by means of surfactants has been studied. Several enzymes (alpha-chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), pyrophosphatase (EC 3.6.1.1), peroxidase (EC 1.11.1.7), lactate dehydrogenase (EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.40)) were used to demonstrate that enzymes can be entrapped into reversed micelles formed by surfactants (Aerosol OT, cetyltrimethylammonium bromide, Brij 56) in an organic solvent (benzene, chloroform, octane, cyclohexane). The enzymes solubilized in this way retain their catalytic activity and substrate specificity. 2. A kinetic theory has been put forward that describes enzymatic reactions occurring in a micelle-solvent pseudobiphasic system. In terms of this theory, an explanation is given for the experimental dependence of the Michaelis-Menten equation parameters on the concentrations of the components of a medium (water, organic solvent, surfactant) and also on the combination of the signs of charges in the substrate molecule and on interphase (++, +-, --). 3. The results obtained by us may prove important for applications of enzymes in organic synthesis and for studying the state and role of water in the structure of biomembranes and active centres of enzymes.

NAD+-dependent hydrogenase from the hydrogen oxidizing bacterium Alcaligenes eutrophus Z1. Stabilization against temperature and urea induced inactivation.

Chemical modification of the NAD+-dependent hydrogenase from the hydrogen oxidizing bacterium Alcaligenes eutrophus Z1 results in considerable enzyme stabilization towards urea and temperature induced inactivation. The stabilizing effect was shown to originate from the suppression of hydrogenase tetramer dissociation. The magnitudes of the stabilizing effects (5-fold and more) were in agreement with the values predicted on the basis of the enzyme thermoinactivation mechanism postulated earlier. Hydrophobic interactions are considered to be critical for the stability of the enzyme quaternary structure. Various methods of hydrogenase immobilization were tested. The enzyme was immobilized with a high retention of activity on aminated silochrom via its carboxylic groups.

Reliability of elements and systems in enzymology.

The applications of the elements of reliability to the description of the stability and aging processes of enzymes and polyenzyme systems have been considered. The inactivation of individual enzymes or polyenzyme systems whose activity varies on the molecular level according to the "all-or-nothing" principle is described by an exponential reliability law. Systems of enzymes which are inactivated according to an exponential law have been considered. It has been shown that in the case of enzymes functioning in parallel, the value of the mean time of continuous operation is determined by the most stable enzyme, while in the case of sequentially functioning enzymes, this value is determined by the most labile. Systems whose aging process is related to the accumulation of "latent errors" and whose inactivation is characterized by gradual breakdowns are described by a normal reliability law. Systems with a combination of exponential and normal reliability laws have been considered. The use of a series of rigid quantitative criteria, such as the mean time for continuous functioning has been proposed for characterizing the stability of enzymes and polyenzyme systems. The theoretical points have been illustrated by experimental data on the stability of enzymes with hydrogenase activities, isolated chloroplasts, and cells of microscopic blue-green algae.

[Kinetic patterns of reactions with immobilized enzymes in open systems]. / Kineticheskie zakonomernosti reaktsii s immobilizovannymi fermentami v otkrytykh sistemakh

For studying the properties of immobilized enzyme, a flow reactor is suggested which provides ideal mixing of the reaction components with the granules of immobilized enzyme. To elucidate the mechanism of the reaction and to determine the effective values of maximal rates and Michaelis constants, an analysis of formal kinetic relationships describing the behaviour of the reactor in the stationary and non-stationary regimes has been carried out. The non-stationary kinetics is analyzed both for the case of step-wise and continuous supply of the substrate. A number of equations has been evolved which describe the time dependences of the concentrations of the substrate and reaction products; relationships have been found allowing determination of maximal rates and Michaelis constants from experimental data.

[Kinetics of reactions in polyenzyme systems. III. Electron transport processes]. / Kinetika reaktsii v polifermentnykh sistemakh. III. Elektronotransportnye protsessy.

Kinetic relationships of electron transport processes in biological systems have been analysed. Formal-kinetic description and discrimination of the two main models of electron transport processes in systems of homogeneously distributed carriers and in systems of structurally bound carriers is presented including steady-state and non-steady-state reaction kinetics. Methods of determination of limit-stage rate constant and rate constants for all elementary steps of the reaction are given. Dependencies of the degree of electron carriers reduction on donor and acceptor concentrations and the influence of reduction or oxidation of carriers on the kinetic properties of electron transport chain (electron influence factor) in the system of structurally-bound carriers have been analyzed. On the basis of kinetic models a molecular-kinetic interpretation of activation effect of mitochondrial respiratory chain during the process of electron transport is offered.

[Kinetics and mechanism of inactivation of isolated chloroplasts]. / Kinetika i mekhanizm inaktivatsii izolirovannykh khloroplastov

A detailed kinetic analysis has been performed of a multistep inactivation of chloroplasts. The kinetic model suggested involves the formation of chloroplast forms differing in stability and activity. A comparison of the kinetic model with the experimental data shows that the mechanism of inactivation of isolated pea chloroplasts consists of at least two forms displaying different activity in the Hill reaction and different stability in solution. The effect was studied of the nature of the buffer and destruction products on the kinetics of chloroplast inactivation in the process of "ageing". In phosphate buffer, where the concentration of phosphate exceeds 40 mM, the effect of the destruction products of chloroplasts on their inactivation is insignificant. The pH dependence on the inactivation kinetics suggests that the pH region from 6 to 9 affects only the rapid kinetic process resulting in the increase in the chloroplast activity; irreversible inactivation of chloroplasts is pH-independent. The temperature dependence of the irreversible inactivation kinetics has been studied and the activation parametres of this stage have been determined. Possible molecular mechanism of the limiting stages of the inactivation of isolated chloroplasts are discussed, which can explain the kinetic data obtained.

[Kinetic patterns of bacterial hydrogenase inactivation]. / Kineticheskie zakonomernosti inaktivatsii bakterial'nykh gidrogenaz

Kinetics of inactivation of hydrogenases is exemplified with the enzyme from Chloropseudomonas ethylica. The effect of gaseous phase, temperature, pH on the inactivation kinetics has been studied. Inactivation of the hydrogenase during incubation of an enzyme solution in air has been studied. Analysis of the kinetic data allowed the conclusion to be made that inactivation involves two forms of the enzyme which differ in their activity and resistance to inactivating actions. Stabilities of the mean time of operation of hydrogenases from Chloropseudomonas ethylica and Thiocapsa roseopersicina are compared. The mechanism of inactivation of hydrogenases from the two sources were found to be similar in many ways.

[Beta-galactosidase from E. coli as a marker in immunoenzyme analysis]. / Beta-galaktozidaza E. coli kak marker v immunofermentnom analize.

In the present review the authors analyze factors influencing sensitivity of the enzyme immunoassay (EIA). beta-Galactosidase from E. coli was chosen as a marker enzyme. Physico-chemical properties of the enzyme, detection methods and various ways of obtaining chemical conjugates with antigens and antibodies are discussed. Some examples of using beta-galactosidase as a label in homogeneous and heterogeneous EIA are given which enables different compounds to be analyzed with a high sensitivity. New approaches employing gene engineering for constructing "fusions" between beta-galactosidase and antigens (alternative to chemical conjugates) are discussed that can be used in the near future.