Effect of small carbohydrates on the catalytic activity of a protease and two glycohydrolases

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.