Operational stability of copolymerized enzymes at elevated temperatures.

The copolymerization method of immobilization was used to obtain preparations of enzymes covalently incorporated in polyacrylamide gel. They possess properties making them suitable for practical use. First, the preparations are hundreds of times more stable against irreversible thermoinactivation than native enzymes. Second, on immobilization, the reversible conformational changes which also lower enzyme activity at elevated temperatures are completely suppressed. As a result, the temperatures of maximum activity for trypsin and alpha-chymotrypsin covalently entrapped in polyacrylamide gel are 75 and 70 degrees C, respectively-25 and 30 degrees C higher than the corresponding values for the native enzymes. Therefore, the copolymerized enzyme preparations have a high operational stability at elevated temperatures.

Electrostatic effects in trypsin reactions. Influence of salts.

The influence of inorganic salts on trypsin-catalyzed reactions has been studied. It is shown that: (a) monovalent cations are reversible competitive inhibitors of tryptic hydrolysis of cationic substrates, whereas their binding has no effect on the reaction of neutral substrates; (b) a nonelectrostatic salt effect on the binding of both cationic and non-ionic substrates is caused by changes in the thermodynamic activity coefficient of the substrate; (c) the rate of trypsin active-site acylation is not affected by inorganic salts with monovalent cations. The data suggest that low-molecular-mass substrates are extracted into the enzyme microphase during substrate binding and further chemical transformations proceed without an access from surrounding medium. It is proposed that formation of a properly oriented dipole in the trypsin binding pocket by the cationic group of the substrate and Asp189 carboxyl is responsible for the elevated acylation rate of trypsin active site by substrates containing lysine and arginine. Introduction of additional negative charges into the enzyme molecule by chemical modification of lysyl residues by pyromellitic anhydride increased the specificity of trypsin towards cationic substrates and inhibitors. Lysine residues are therefore considered as suitable targets for site-directed mutagenesis aimed at the improvement of selectivity and catalytic properties of trypsin.

High stability to irreversible inactivation at elevated temperatures of enzymes covalently modified by hydrophilic reagents: alpha-Chymotrypsin.

Based on the idea that proteins can be stabilized by a decrease in the thermodynamically unfavorable contact of the hydrophobic surface clusters with water, alpha-chymotrypsin (CT) was acylated with carboxylic acid anhydrides or reductively alkylated with aliphatic aldehydes. Modification of CT with hydrophilic reagents leads to 100-1000-fold increase in stability against the irreversible thermoinactivation. The correlation holds: the greater the hydrophilization increment brought about by the modification, the higher is the protein thermostability. After some limiting value, however, a further increase in hydrophilicity does not change thermostability.It follows from the dependence of the thermoinactivation rate constants on temperature that for hydrophilized CT there is the conformational transition at 55-65 degrees C into an unfolded state in which inactivation is much slower than that of the low-temperature conformation. The thermodynamic analysis and fluorescent spectral data confirm that the slow inactivation of hydrophilized CT at high temperatures proceeds via a chemical mechanism rather than Incorrect refolding operative for both the native and low-temperature form of the modified enzyme. Hence, the hydrophilization stabilizes the unfolded high-temperature conformation by eliminating the incorrect refolding.

Reversible conformational transition gives rise to 'zig-zag' temperature dependence of the rate constant of irreversible thermoinactivation of enzymes.

We have obtained unusual 'zig-zag' temperature dependencies of the rate constant of irreversible thermoinactivation (k(in)) of enzymes (alpha-chymotrypsin, covalently modified alpha-chymotrypsin, and ribonuclease) in a plot of log k(in) versus reciprocal temperature (Arrhenius plot). These dependencies are characterized by the presence of both ascending and descending linear portions which have positive and negative values of the effective activation energy (Ea), respectively. A kinetic scheme has been suggested that fits best for a description of these zig-zag dependencies. A key element of this scheme is the temperature-dependent reversible conformational transition of enzyme from the 'low-temperature' native state to a 'high-temperature' denatured form; the latter form is significantly more stable against irreversible thermoinactivation than the native enzyme. A possible explanation for a difference in thermal stabilities is that low-temperature and high-temperature forms are inactivated according to different mechanisms. Existence of the suggested conformational transition was proved by the methods of fluorescence spectroscopy and differential scanning calorimetry. The values of delta H and delta S for this transition, determined from calorimetric experiments, are highly positive; this fact underlies a conclusion that this heat-induced transition is caused by an unfolding of the protein molecule. Surprisingly, in the unfolded high-temperature conformation, alpha-chymotrypsin has a pronounced proteolytic activity, although this activity is much smaller than that of the native enzyme.

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.