Kinetic characterization of the neutral protease vimelysin from Vibrio sp. T1800.
The kinetics of the hydrolysis of dipeptide and tripeptide substrates by the recently discovered neutral protease from Vibrio species T1800 (vimelysin) were studied. In the pH dependence of the apparent second-order rate constant, the pKa2 value of vimelysin (approximately 6.5) was significantly lower than thermolysin (8.3), although the pKa1 (approximately 5.1) values were comparable (5.0). The Kcat/Km(lim) parameter for hydrolysis of Fua-Gly-PheNH2 (Fua = furylacryloyl) was more than sevenfold greater than for Fua-Gly-LeuNH2. This higher specificity for Fua-Gly-PheNH2 was deduced for both Kcat and Km parameters. Fua-Phe-PheNH2 showed the highest Kcat/Km(app) value of the six substrates studied. The discrimination between Phe/Leu at the P1' site was most evident when the P1 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, 463-467], the Arrhenius plot of the apparent second-order rate constant for the hydrolysis of Fua-Gly-LeuNH2 showed an inverse temperature dependence; higher reaction rates were observed at lower temperatures. This was not merely due to the pKa shift nor to thermal denaturation of the enzyme coupling, but rather to the Kcat(app) 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.
Kunugi
S
S
Department of Polymer Science and Engineering, Kyoto Institute of Technology, Japan.
Koyasu
A
A
Kitayaki
M
M
Takahashi
S
S
Oda
K
K
D002942:Circular Dichroism; D004151:Dipeptides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013696:Temperature; D014733:Vibrio / Q000201:enzymology*
D004151:Dipeptides; D009842:Oligopeptides; D008666:Metalloendopeptidases; C099068:vimelysin