Additional data about thermolysin specificity in buffer- and glycerol-containing media

Université de Technologie de Compiègne, Compiègne, Picardie, France
Biochimica et Biophysica Acta (Impact Factor: 4.66). 02/1997; 1337(1):143-8. DOI: 10.1016/S0167-4838(96)00142-2
Source: PubMed


Synthesis and use of various substrates permit an improved approach to thermolysin-peptide recognition and elucidation of several new criteria affecting enzyme specificity. Nature and position of the recognized residue, role of adjacent amino acids, lateral chain hydrophobicity, and volume and length of peptides were all considered. Hydrolysis reactions were also carried out in the presence of glycerol; the effect of microenvironment modifications was quantitative, for example in inducing variations in catalytic reaction rates, and also qualitative, such as in influencing affinity.

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    • "On the other hand, aqueous solutions of polyols, as additives, are currently used to study thermostabilization of enzymes at concentrations lower than 40% [4 – 6]. In recent work, 73% polyol concentration was used to study enzyme specificity [7]. However, in most of the cases at high cosolvent concentration, aggregates and/or precipitates of proteins in buffer were observed [5] [8]. "
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    ABSTRACT: UV scanning of alpha-chymotrypsin dissolved in neat glycerol and water showed no significant differences in its spectra at pH 7.8. Fluorescence scanning revealed a strong dependence on pH values (between 5.9 to 10.5) of the maximum wavelength emission in water and no pH-dependence in 99% glycerol supplemented with 1% of appropriate buffers. The profile of alpha-chymotrypsin activity dissolved in water-glycerol mixtures with phenyl acetate as substrate displayed two maximum: highest peak was found at 100% water, and the second one was observed in 99% glycerol concentration with about 40% of the relative activity. Optimum pH of the soluble alpha-chymotrypsin in glycerol showed a displacement of 1 pH/U towards the alkaline side compared to water at pH 8.0. Kinetic and thermodynamic analysis using kinetic measurements of the thermal stability of alpha-chymotrypsin showed a higher inactivation rate in neat glycerol as compared to water in 30 to 45 degrees C range, however, when temperature increases enzyme stability in glycerol is better than water. Thermostability of trypsin and alpha-chymotrypsin dissolved in glycerol at 100 degrees C showed a half reaction time of approximately 7 and 20 h, respectively, and less than 1 minute in aqueous buffer for both enzymes.
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    ABSTRACT: Summary The conformations of thermolysin synthetic substrates in H2O/D2O (9/1) and glycerol-d 5 (5 M) are investigated using two-dimensional nuclear magnetic resonance spectroscopy and molecular modeling. The structures obtained from molecular modeling and NMR studies are compared. Comparisons of these structures with bound inhibitor in the active site of thermolysin are also discussed.
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    ABSTRACT: We have examined the proteolysis of model proteins by thermolysin when dissolved in aqueous buffer at neutral pH in the presence of 50% (by vol.) trifluoroethanol (TFE). Under these solvent conditions, proteins acquire a new conformational state characterized by enhanced helical secondary structure, but lacking the specific tertiary interactions of the native species. It was found that the TFE-state of proteins dictates very selective peptide bond fissions by the TFE-resistant thermolysin, which otherwise shows broad substrate specificity. Nicked protein species with a single peptide bond hydrolyzed have been prepared and isolated to homogeneity in the case of bovine ribonuclease A (cleavage at Asn34-Leu35), hen lysozyme (Lys97-Ile98), bovine α-lactalbumin (Ala40-Ile41) and horse cytochrome c (Gly56-Ile57).
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