Pressure Stability of Low and High Temperature Acitive Metalloproteases
Shigeru KUNUGI*a and Kouhei ODAb
a Department of Polymer Science and Technology and
b Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto, 606, JAPAN
Two types of metalloproteases from microbial origins, thermolysin from B. thermoproteolyticus and vimelysin from Vibrio Sp., were studied for their pressure and temperature stability and activity. These two enzymes show rather contrastive temperature dependences in their catalytic activity. Thermolysin, as isolated from a thermophilic bacteria, is known to show moderate temperature resistance (1), while vimelyisin, isolated by Oda et al. from rather low temperature-adapted bacteria (2), shows apparently inversed Arrhenius plot; vimelysin shows much higher activity at 15℃ than 35℃ (3).
As for pressure dependence, both enzymes show apparent pressure-activation of the hydrolytic catalysis of synthetic peptide substrates. Thermolysin showed more than 25-fold higher activity at 150 MPa (4,5) and futher increase in pressure results in decrease in the activity. The extent of pressure-activation for vimelysin is smaller than thermolysin and showed around 5-fold higher kcat/Km at 150 MPa, which resembles to the result of a mesophilic metalloprotease, one from B. subtilis var. amyloliquifaciens (4). The activation volumes calculated from kcat/Km were about 60 ml/mol for thermolysin and about 30 ml/mol for vimelysin.
Pressure-temperature diagrams of these enzymes with respect to the catalytic activity and fluorescence intensity of intrinsic tryptophans were drawn. The apparent (catalytic) optimum conditions for thermolysin were found to be 60℃ and 200 MPa and those for vimelysin were 15℃ and 150 MPa for acyl-dipeptideamide substrate. Intrinsic fluorescence showed that thermolysin undergoes structural change above 300 MPa with a negative expansibility (-0.45 ml/mol/K). Vimelysin shows a transition in fluorescence above 250 MPa at 30℃, but the transition pressure becomes lower in both higher and lower temperature sides.
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