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

Laboratoire de Technologie Enzymatique, URA 1442 CNRS, Compiègne University, France.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 02/1997; 1337(1):143-8.
Source: PubMed

ABSTRACT 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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    ABSTRACT: The effect of sugars and polyols on the kinetic behavior of a protease, thermolysin from Bacillus thermoproteolyticus, and two glycohydrolases, pullulanase from Bacillus sp. 3183 and inulinase from Aspergillus niger was studied. Kinetic parameters, especially the catalytic efficiency (kcat/kM) of enzymes were determined in buffer and in the presence of small carbohydrate additives. It was shown that the catalytic efficiencies of the studied enzymes depend on both the nature and the concentration of the additive. Thermolysin was found to be more activated by sugars and polyols than pullulanase or inulinase. This implies a specific effect of the additive besides its effect on the enzyme micro-environment.Activation free energy of the thermolysin catalyzed reaction in the presence of additives was found to be lower than in buffer medium. As temperature was kept constant, the activation energy change is assignable to entropic change. Such an effect, mainly responsible for the activation of the enzyme by the micro-environment, is called solvactivation. In turn, the lowering of catalytic efficiency (below the level obtained with buffer) by such additives as glycerol is called solvinhibition.An infrared spectroscopy study of thermolysin in the presence of additives showed that no conformational change is observed and that small carbohydrates contribute to preferential hydration of the enzyme. Raman spectra showed that no direct interaction of thermolysin was observed in the presence of glycerol and that the secondary structure of the enzyme remained unchanged.It was possible to differentiate the effects of small carbohydrates on the studied enzymes. The specific activating or inhibiting effect seems linked to the effect of these solutes on the water structure.
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    ABSTRACT: The catalytic activity of thermolysin during the hydrolysis of n-(3-[2-furyl]acryloyl)-Gly-Leu amide is noticeably enhanced in the presence of sugars and polyols. A series of polyhydroxylic additives were tested and the degree of activation was found to depend on both the concentration and nature of the additive. Sucrose and trehalose were found to yield the higher activation effect whereas glycerol was found to yield an inhibition of thermolysin in a large domain of concentration. A mechanism of activation based on the lowering of the energy barrier for the enzymatic reaction is proposed. This free energy barrier lowering is very likely due to water structure modification by the additives.
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