title: 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.
authors.affiliation: Department of Polymer Science and Engineering, Kyoto Institute of Technology, Japan. kunugi@ipc.kit.ac.jp
authors.forename: S
authors.initials: S
authors.lastname: Kunugi
authors.forename: M
authors.initials: M
authors.lastname: Kitayaki
authors.forename: Y
authors.initials: Y
authors.lastname: Yanagi
authors.forename: N
authors.initials: N
authors.lastname: Tanaka
authors.forename: R
authors.initials: R
authors.lastname: Lange
authors.forename: C
authors.initials: C
authors.lastname: Balny
mesh_terms: D001671:Biochemistry / Q000295:instrumentation / Q000379:methods; D002942:Circular Dichroism; D006207:Half-Life; D011312:Pressure; D013057:Spectrum Analysis / Q000379:methods; D013696:Temperature; D013820:Thermolysin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D013820:Thermolysin