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V V Mozhaev

Publications and source records attributed to V V Mozhaev.

35 records · Page 2Linked to original sources
PubMed1989

Catalytic activity and denaturation of enzymes in water/organic cosolvent mixtures. Alpha-chymotrypsin and laccase in mixed water/alcohol, water/glycol and water/formamide solvents.

The dependence of the catalytic activities of alpha-chymotrypsin and laccase on the concentration of organic cosolvents (alcohols, glycols and formamides) in mixed aqueous media has a pronounced threshold character: it does not change up to a critical concentration of the non-aqueous cosolvents added, yet further increase of the latter (by only a small percentage, by vol.) leads to an abrupt decrease in enzyme activity. Fluorescence studies indicate that the inactivation results from reversible conformational changes (denaturation) of the enzymes. There is a linear correlation between the critical concentration of residual water (at which the enzyme inactivation occurs in a threshold manner) and the hydrophobicity of the organic cosolvents added. A quantitative criterion is suggested for the selection of organic cosolvents to be used for enzymatic reactions in homogeneous water/organic solvent media.

Alcohols↗
PubMed1988

Protein stabilization via hydrophilization. Covalent modification of trypsin and alpha-chymotrypsin.

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. Alpha-chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of alpha-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 degrees C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.

Acylation↗
PubMed1988

Structure-stability relationship in proteins: fundamental tasks and strategy for the development of stabilized enzyme catalysts for biotechnology.

The problem of relationships between the protein structure and its stability comprises two major questions. First, how to elucidate the peculiarities of the protein structure responsible for its stability. Second, knowing the general molecular basis of protein stability, how to change the structure of a given protein in order to increase its stability. This review is an attempt to show the modern state of the first (fundamental) and the second (applied) aspects of the problem.

Bacterial Proteins↗
PubMed1981

The influence of effectors on the refolding (reactivation) of immobilized trypsin.

The refolding of immobilized trypsin in the presence of various effectors of its enzymatic activity has been studied. Trypsin covalently bound to Sephadex G-200 was made to unfold in a concentrated solution of urea; at the same time its S-S bonds were split with the help of dithiothreitol. The preparation was then separated from the splitting agents, and one of the effectors of the enzymatic activity of trypsin (boric acid, benzamidine, pancreatic or soybean inhibitors of trypsin, N-benzoyl-L-arginine ethyl ester, and N-tosyl-L-arginine methyl ester) was added and the reactivation of immobilized enzyme was studied in the absence of catalysts of thiol-disulphide exchange. The following effects were found. 1. The reactivation of immobilized trypsin in the presence of specific substrates or protein inhibitors proceeds with the same yield (2-5%) as in their absence. 2. In the presence of benzamidine or boric acid (competitive inhibitors) the reactivation yields of the immobilized trypsin increased 5-fold and 12-fold respectively, and became equal to 15% and 40%. Comparison of these results with the statistical probability of formation of the six native S-S bonds from twelve SH groups (approximately 0.01%) shows that if trypsin is made to refold in an immobilized state in the presence of a 'good' effector, the yield of the reactivation of the enzyme can be increased several thousand times. 3. Similar effects were observed for trypsin immobilized on Sepharose 4B. A model is suggested in terms of which the influence of various effectors on trypsin refolding is explained as being a result of their ability to bind with an intermediate folded forms of protein followed by a shift of the equilibrium towards 'regular' conformers.

Catalysis↗
PubMed1980

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.

Chymotrypsin↗
PubMedDate not supplied

[Regulation of thermal stability of enzymes by changing the composition of media. Native and modified alpha-chymotrypsin].

Stabilizing effect of denaturing salts on irreversible thermoinactivation of native and modified alpha-chymotrypsin at elevated temperatures is observed. The effect is caused by a shift of conformational equilibrium, at the primary step of reversible unfolding in the course of thermoinactivation, to a more unfolded form which is not able to refold "incorrectly". The stability of alpha-chymotrypsin is regulated within a wide range by medium alteration: the stabilizing effects are similar to those achieved by multipoint attachment of the enzyme to a support or by hydrophilization of protein by covalent modification.

Chymotrypsin↗
PubMedDate not supplied

[Correlation of enzyme thermostability and its surface hydrophobicity (using a modified alpha-chymotrypsin as an example)].

Basing on the hypothesis that contact of hydrophobic surface clusters of proteins with water is thermodynamically disadvantageous, it is suggested to carry out the hydrophilization of protein surface by covalent modification in order to increase its thermostability. Hydrophilic fragments were introduced into the surface of alpha-chymotrypsin using acylation by anhydrides of aromatic carboxylic acids and reductive alkylation by aliphatic aldehydes. As a result of the hydrophilization the stability of the enzyme against irreversible thermoinactivation increased thousand-fold. The correlation is observed between the degree of hydrophilization of the protein surface and the increase in thermostability of modified alpha-chymotrypsin. The level of thermostability achieved by covalent modification of alpha-chymotrypsin is practically equal to thermostability of proteinases from extreme thermophiles, the most stable proteolytic enzymes currently known.

Binding Sites↗
PubMedDate not supplied

[Inactivation and reactivation of proteins (enzymes)].

Numerous examples of protein inactivation are considered. Several principal inactivation mechanisms are distinguished: aggregation, thiol-disulfide exchange, alteration in the protein primary structure, dissociation of cofactor molecules from the enzyme active site, dissociation of proteins with quaternary structure into subunits, and conformational changes. All the mechanisms interweave during protein inactivation. In many cases, however, conformational transformations in protein play an important role; they accompany and often trigger other mechanisms of inactivation which would be impossible without preliminary alterations in protein structure. The published data on reactivation of inactivated proteins are considered. Reactivation was a success when inactivation was caused by aggregation, chemical modification of SH-groups of S-S bonds, or resulted from "irreversible" conformational changes.

Amino Acid Sequence↗