Activity and stability of a neutral protease from Vibrio sp. (vimelysin) in a pressure-temperature gradient.
The apparent second-order rate constant of hydrolysis of Fua-Gly-LeuNH2 by vimelysin, a neutral protease from Vibrio sp. T1800, was measured in a variable pressure-temperature gradient (0. 1-400 MPa and 5-40 degrees C). The apparent maximum rate was observed at approximately 15 degrees C and 150-200 MPa; the pressure-activation ratio (kcat/Km(max)/kcat/Km(0.1 MPa)) was reached about sevenfold. The pressure dependence of the kcat and Km parameters at constant temperature (25 degrees C) revealed that the pressure-activation below 200 MPa was mainly caused by a change in the kcat parameter. The change in the intrinsic fluorescence intensity of vimelysin was also measured in a pressure-temperature plane (0.1-400 MPa and -20 to +60 degrees C). The fluorescence intensity was found to decrease by increasing pressure and temperature, and the isointensity contours were more or less circular. The tangential lines to the contours at high temperatures and low to medium pressures seem to have slightly positive slopes, which was reflected by the higher residual activities left after incubations at higher temperatures and medium pressure (200 MPa and 50 degrees C) and by the almost intact secondary structure left after 1 h of incubation at 200 MPa and 40 degrees C, as studied by circular dichroism. These results were compared with the corresponding results for thermolysin, a moderately thermostable protease from Bacillus thermoproteolyticus. Apparent differences that might be related to the temperature adaptations of the respective source microbes are also discussed.
Ikeuchi
H
H
Laboratory for Biopolymer Chemistry, Department of Polymer Science, Kyoto Institute of Technology, Japan.
Kunugi
S
S
Oda
K
K
D001407:Bacillus / Q000201:enzymology; D002942:Circular Dichroism; D004151:Dipeptides / Q000378:metabolism; D004795:Enzyme Stability; D005453:Fluorescence; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D011312:Pressure; D017433:Protein Structure, Secondary; D013696:Temperature; D013816:Thermodynamics; D013820:Thermolysin / Q000737:chemistry / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology*
D004151:Dipeptides; D008666:Metalloendopeptidases; C099068:vimelysin; D013820:Thermolysin