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-' at 25°C. A v i was accompanied by a negative activation expansibility and a value of -95 ml mol-' was obtained at 45 "C. A prolonged incubation of thermolysin under high pressure, however, caused a timedependent 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.Keywords: thermolysin ; high pressure; activation ; denaturation ; fourth-derivative spectrum.Thermolysin is a thermostable microbial neutral protease containing zinc as a cofactor [l]. This enzyme has been a target of many studies on its catalytic properties [2-61 and structural aspects 17, 81, since this enzyme has attracted attention with respect to membrane-bound metal endopeptidase [9-I], as well as for its ability to synthesize many useful peptides [12-141. Recently, pressure techniques have been developed as powerful tools for the study of the modulation of enzymatic activity and protein structure, including complexes and aggregates [ 15, 161. These high pressure effects have been exploited in biotechnological and bioengineering applications [ 17).We have studied some of the kinetic aspects of thermolysin, in hydrolytic [18][19][20][21][22] and condensation [23-251 reactions, and found that it shows considerable activation by pressure 1191, which can be utilized for high-pressure enzyme catalysis inducing peptide condensations and protein processing [23, 261. During a series of studies, however, we have noticed that the effect of a much higher pressure is different from the effects observed at relatively lower pressure (< 150 MPa). Here, we investigated the effect of a much higher pressure on thermolysin for its catalytic and spectroscopic properties.In this study, we applied two new methods, besides conventional batch-wise reaction monitoring. An important problem for reaction rate measurements under high pressure is encountered A,pr(Dnp), W-(2,4-dinitrophenyl)-l-2,3-diaminopropionyl; Fua-, 3-(2-furyl)acryloyl-; MeOcAc, (7-methoxycoumariu-4-y1)acetyl.Enzyme. Therrnolysin (EC 3.4.24.27).in the mixing or the initiation process of the reaction. Ty...

The effects of high pressure, up to 400 MPa, on the catalytic activity and the fluorescence and CD of carboxypeptidase Y (CPDY) were investigated. CPDY showed a pH-dependent Suc–Ala–Ala–Pro–Phe–pNA hydrolysis similar to other neutral substrates. The apparent second-order rate showed a gradual decrease with increasing pressure, which was related to an increase in Km and a decrease in kcat. The intrinsic fluorescence of CPDY showed a gradual decrease in the intensity and a red shift in the maximum wavelength with pressure. The transition curve did not follow a simple tow-state transition, but contained at least three states. The first transition occurred at around 100 MPa and the second one occurred at pressures higher than 200 MPa. After incubation at 300 MPa, both the peak intensity and the maximum wavelength did not show complete restoration; the pressure-induced change is substantially irreversible. The latter change corresponds to the increased binding of a fluorescent hydrophobic probe molecule (8-anilino-1-naphthalenesulfonic acid) to this protein; however, the CD spectrum showed practically no evidence of irreversible changes in the protein’s secondary structure.

The hydrolysis and peptide formation reactions of carboxypeptidase Y (CPDY) were studied in a ternary system consisting of octyl -D-glucopyranoside ( -OG), water, and octanol (OcOH). This was performed to elucidate the effects of the structures of the surrounding medium on biocatalytic reactions, with special reference to enzyme reactivity and the morphology of the surfactant aggregates. First, the effect of -OG on the catalytic activity of CPDY (hydrolytic reaction) was studied in a surfactant/ water binary system. -OG was found to inhibit CPDY activity for Suc-Ala-Ala-ProPhe-pNA, but somehow accelerated the reaction with Bz-Tyr-pNA. When the CPDY reactions were measured under several conditions in the phase diagram of the ternary system, CPDY was found to be strongly affected by the size and form of the microstructures in each phase. In the reverse micelle (L 2 ) phase, the hydrolytic rate was maximally increased with an increase in the water content; the highest rate was observed at the boundary of the L 2 /2L or L 2 /F (reversed cylindrical diameter) phase. In the aminolysis reaction catalyzed by this enzyme, a higher yield of the dipeptide was also observed near the boundary of the L 2 /2L or L 2 /D + X phase and also in the F phase. The fraction of aminolysis (f a ) was relatively high and rather insensitive to changes in the phase, with the exception that the f a was low in the D/X phase.