Effects of pH, Temperature, and Alcohols on the Remarkable Activation of Thermolysin by Salts.

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  • Inouye Kuniyo
    Department of Food Science and Technology, Faculty of Agriculture, Kyoto University
  • Lee Soo-Bok
    Department of Food Science and Technology, Faculty of Agriculture, Kyoto University
  • Nambu Koji
    Department of Food Science and Technology, Faculty of Agriculture, Kyoto University
  • Tonomura Ben'ichiro
    Department of Food Science and Technology, Faculty of Agriculture, Kyoto University

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  • Effects of pH Temperature and Alcohols

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Abstract

The activity of thermolysin in the hydrolysis of N-[3-(2-furyl) acryloyl] (FA)-dipeptide amides and N-carbobenzoxyl-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced by high concentrations (1-5M) of neutral salts. The activation is due to an increase in the molecular activity, kcat, while the Michaelis constant, Km, is not affected by the addition of NaCl. In the present study, the effect of NaCl on the thermolysin-catalyzed hydrolysis of FA-glycyl-L-leucine amide (FAGLA) has been examined by changing the pH and temperature, and by adding alcohols to the reaction mixture. The enzyme activity, expressed by kcat/Km, is pH-dependent, being controlled by two functional residues with pKa values of 5.4 and 7.8 in the absence of NaCl. The acidic pKa is shifted from 5.4 to 6.7 by the addition of 4M NaCl, while the basic one is not changed. The degree of activation at a given concentration of NaCl is pH dependent in a bell-shaped manner with the optimum pH around 7. Although the activity increases in both the presence and absence of NaCl with increasing temperature from 5 to 35°C, the degree of activation decreases. Alcohols inhibit thermolysin, and the degree of activation decreases with increasing alcohol concentration. The degree of activation tends to increase with increasing dielectric constant of the medium, although it varies considerably depending on the species of alcohol. Electrostatic interactions on the surface and at the active site of thermolysin are suggested to play a significant role in the remarkable activation by salts.

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