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 kinetics of the hydrolysis of dipeptide and tripeptide substrates by the recently discovered neutral protca\e from Vihrio species T1800 (vimelysin) were studied. In the pH dependence of the apparent second-order rate constant, the pK,, value of vimelysin (~6 . 5 ) was significantly lower than thermolysin (8.3 1. although the pK,, (~5 . 1 ) values were comparable (5.0). The kJKn,,,i,,,l parameter for hydrolysis of Fua-Gly-PheNH2 (Fua = furylacryloyl) was more than sevenfold greater than for Fua-Gly-LeuNH,. This higher specificity for Fua-Gly-PheNH, was deduced for both k,,, and K,, parameters. Fua-Phe-PheNH, showed the highest kc,r,lKn,,app) value of the six substrates studied. The discrimination between PheLeu at the PI' site was most evident when the PI site was not sufficiently filled.Reflecting the characteristically high proteolytic activity of vimelysin at lower temperatures [Oda, K., Okayama, K., Okutomi, K., Shimada, M., Sato, R. & Takahashi, S. (1996) Biosci. Biotech. Biochem. 60, 463 -4671, the Arrhenius plot of the apparent second-order rate constant for the hydrolysis of Fua-GlyLeuNH2 showed an inverse temperature dependence ; higher reaction rates were observed at lower temperaliires. This was not merely due to the pK:, shift nor to thermal denaturation of the enzyme coupling, but rather to the kcilr,+,,,) parameter, which alone showed an inverse temperature dependence. A model containing two temperature-dependent forms of the active enzyme was postulated to explain this unique temperature dependence.Keywords : neutral protease ; vimelysin ; metalloprotease ; temperature dependence ; Vibrio.Several metal-containing neutral proteases have been found in various microorganisms. Of these proteases, thermolysin, a thermostable microbial neutral protease [ 11, and its homologous enzymes from Bucillus sp. have been the target of many studies on their catalytic properties [2-61 and structure [7, 81. We have also studied some o l the kinetic aspects of these enzymes [9-161. Metallo-neutral proteases have also attracted much attention, especially with the relation to membrane-bound metal-containing endopeptidases [17-191, and the structural and mechanistic aspects of inhibitor interactions have been studied 120- 241.Recently, Oda et al. isolated and purified a neutral protease from Vibrio sp. T1800 (the species is not specified yet) [25]. They found that this enzyme, named vimelysin, has no similaritiy in its N-terminal sequence with metallo-endoproteases from Bacillus sp. and is considerably different in its catalytic properties. It showed high proteolytic activity at lower temperatures and had characteristic specificities for some natural polypeptides [26].In the Vibrio species, several alkaline proteases have been isolated; some of these enzymes have been reported to show some of the characteristics of metallo-proteases [25 -301.Vimelysin has considerable similarity in its N-terminal sequence (1-20) with these enzymes [25, 31. 321. However,Correspu~idence to S. Kunugi,

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.

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ABSTRACT:The relationship of the salt-activation of thermolysin and the thermodynamic properties was investigated by using DSC and high-pressure spectrofluorometry. As previously reported [Holmquist and Vallee, Biochemistry, 15, 101 (1976), Fukuda and Kunugi, Biocatalysis, 2, 225 (1989) showed monotonous increase in the activity, and the rankings of rate increase at 2 M were NaIn the presence of salt, thermolysin showed lower peak temperature in DSC, and the instability was in the order of SCNBy addition of 0.5 M NaBr, the contour map of the peak intensity of thermolysin intrinsic fluorescence became much simpler, and the contour shifted to lower temperature. The ÀÁV values of the transition decreased with increasing salt at lower concentration range, but then increased at higher concentration, especially at higher temperature. Thus ÁV became more negative with increasing temperature. These results are discussed with respect to the ion distribution among the lowand high-density water phases in the protein solution, and the surface charge, hydration, and flexibility of enzyme protein, especially at the transition state of the catalysis. [doi:10.1295/polymj.PJ2006106] KEY WORDS Thermolysin / Salt Effects / High Pressure / Protein / Denaturation / Calorimetry / Fluorescence / Specific functions performed by protein is realized by its hierarchical structures. A change or a collapse of the higher-order structure results in a loss of the function. Thus stability of proteins and enzymes is the main concern of the researchers on proteins in various fields, from very fundamental to industrial or clinical applications.