Authors: Kunugi S.¹; Koyasu A.¹; Kitayaki M.¹; Takahashi S.²; Oda K.²

Source: FEBS Journal, Volume 241, Number 2, October 1996 , pp. 368-373(6)

Publisher: Wiley-Blackwell

Abstract:

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 pK_a2 value of vimelysin (6.5) was significantly lower than thermolysin (8.3). although the pK_a2 ( 5.1) values were comparable (5.0). The k_cat/K_m(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 k_cat and K_m parameters. Fua-Phe-PheNH₂ showed the highest k_cat/K_m(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 pK_a shift nor to thermal denaturation of the enzyme coupling, but rather to the k_cat(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.

Keywords: neutral protease; vimelysin; metalloprotease; temperature dependence; Vibrio

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1432-1033.1996.00368.x

Affiliations: 1: Laboratory for Biopolymer Physics, Department of Polymer Science and Engineering 2: Department of Applied Biology, Kyoto Institute of Technology, Japan

Publication date: 1996-10-01

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