Open AccessThe Effect of High Pressure on Thermolysin
Author(s) -
Kunugi Shigeru,
Kitayaki Moto,
Yanagi Yuuichi,
Tanaka Naoki,
Lange Reinhard,
Balny Claude
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.t01-1-00567.x
Subject(s) - thermolysin , chemistry , substrate (aquarium) , absorbance , circular dichroism , conformational change , amide , volume (thermodynamics) , analytical chemistry (journal) , stereochemistry , chromatography , biochemistry , enzyme , thermodynamics , oceanography , trypsin , physics , geology
The effects of high pressure on thermolysin activity and spectroscopic properties were studied. Thermolysin showed distinct pressure‐induced activation with a maximum observed at 200–250 MPa for a dipeptide amide substrate and at 100–120 MPa for a heptapeptide substrate. By examining the pressure dependence of the hydrolytic rate for the former substrate using a high pressure stopped‐flow apparatus as a mixing device under elevated pressures, the activation volume of the reaction was ‐71 ml mol −1 at 25°C. ΔV‡ was accompanied by a negative activation expansibility and a value of ‐95 ml mol −1 was obtained at 45°C. A prolonged incubation of thermolysin under high pressure, however, caused a time‐dependent deactivation. These changes due to pressure were monitored by several spectroscopic methods. The fourth‐derivative absorbance spectrum showed an irreversible change, mostly in the tyrosine and tryptophan regions, at a pressure higher than 300 MPa. Intrinsic fluorescence and circular dichroism measurements of thermolysin in solution also detected irreversible changes. All these measurements indicated that a change occurred at higher pressures and are explained by a simple two‐state transition model accompanied by a large, negative change in the volume of reaction.