Effects of pressure on the activity and spectroscopic properties of carboxyl proteinases. Apparent correlation of pepstatin-insensitivity and pressure response.
The pressure dependence of the activity and spectroscopic properties of four carboxyl proteinases were investigated. Two were pepstatin-sensitive carboxyl proteinases (porcine pepsin and proteinase A from baker's yeast) and two were pepstatin-insensitive carboxyl proteinases (from Pseudomonas sp. 101 (pseudomonapepsin; PCP) and Xanthomonas sp. T-22 (xanthomonapepsin; XCP)). The specificity constant [k(cat)/K(m(app))] of PCP and XCP for a synthetic peptide substrate showed only a slight decrease with increasing pressure, whereas pepsin and proteinase A showed substantial disactivation at higher pressures. The calculated apparent activation volume (Delta V((k(cat)/(K(m)) was about 1, 3, 13, and 14 mL.mol(-1) for PCP, XCP, pepsin, and proteinase A, respectively. The hydrolysis of acid-denatured myoglobin by the four carboxyl proteinases was only slightly affected by high pressure (except for proteinase A at 400 MPa), in contrast to the results for the peptide hydrolysis. In fact, PCP, XCP, and proteinase A actually showed slightly higher degradations of acid-denatured myoglobin at higher pressures. The residual activities of these enzymes after the incubation at high pressures implied a pressure-induced stabilization towards autolysis. The changes in the fourth derivative near-UV absorbance spectrum of the four enzymes in aqueous solution were measured at various pressures from 0.1 to 400 MPa. Upon an increase in pressure, the peaks from PCP and XCP red-shifted slightly, whereas pepsin and proteinase A blue-shifted substantially, thus indicating a more polar environment. The intrinsic fluorescence also decreased upon increasing pressure. However, the change for XCP was rather small, but the change for the other three was very large. The changes in the peak wavelength for pepsin and proteinase A were characteristic, and also indicated a more polar environment under high pressure. An analysis by the center of spectra mass (CSM) gave the Delta G and Delta V of transition as 9.8 kJ x mol(-1) and -24 mL x mol(-1) (pepsin) and 11.7 kJ x mol(-1) and -43 mL x mol(-1) (proteinase A), respectively, by assuming a simple two-state transition. The circular dichroism (CD) showed relatively small changes after 1-h incubations at 400 MPa, indicating that the secondary structures were largely maintained.
Fujiwara
S
S
Laboratory for Biopolymer Chemistry, Department of Polymer Science and Engineering, Kyoto Institute of Technology, Japan.
Kunugi
S
S
Oyama
H
H
Oda
K
K
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010434:Pepsin A / Q000737:chemistry; D010436:Pepstatins / Q000378:metabolism*; D011312:Pressure; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013552:Swine; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D014466:Ultraviolet Rays; D014974:Xanthomonas / Q000201:enzymology
D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D014443:Tyrosine; D014364:Tryptophan; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A; C022442:aspergillopepsin II; C031375:pepstatin