The effect of high pressure on thermolysin.

Abstract

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 degrees C. Delta V++ was accompanied by a negative activation expansibility and a value of -95 ml mol(-1) was obtained at 45 degrees 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.