Kinetic Characterization of the Neutral Protease Vimelysin from Vibrio Sp. T1800

Abstract

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,

Exploring the subsite‐structure of vimelysin and thermolysin using FRETS‐libraries

“…T1800 discovered by K. Oda et al in 1996, is a unique enzyme characterized by its high activity at low temperature and its resistance to organic solvents [1][2][3][4]. Recently, we cloned and sequenced a gene encoding vimelysin (ORF: 1821 bp) [5].…”

Substrate Specificity in the Highly Heterogeneous M4 Peptidase Family Is Determined by a Small Subset of Amino Acids

“…The availability of an impressive amount of sequence, structural, and kinetic data renders this group of proteases an ideal subject for rational design strategies. Although some of the family members have been characterized individually (5,(21)(22)(23)(24), a consistent comparison with an identical substrate set and a uniform set of assay conditions has never been conducted. Previously it was suggested that TLPs exhibit a preference for large hydrophobic P 1 Ј residues (Leu or Phe) (1,17,21,22).…”

Intestinal Resection- and Steroid-Associated Alterations in Gene Expression Were Not Accompanied by Changes in Lipid Uptake

“…There are several LBP in the intestine [13, 29, 30, 31, 32], but L-FABP is unlikely to play a major role, as suggested by the marked decline in jejunal L-FABP mRNA, but no alteration in lipid uptake (table 5). Recent support for the uncoupling between intestinal lipid uptake and LBP comes from the findings that (1) clones of mice with a disrupted gene for the intestinal FABP (I-FABP) are viable, have elevated plasma triacylglycerol, and weigh more than the wild-type littermates [56], and (2) dietary and diabetic alterations in lipid uptake are not paralleled by similar alterations in LBP expression [57].…”

“…In this study we did not measure BBM lipids, so we are not able to comment upon whether the absence of an effect of steroids on fatty acid uptake was due to their lack of effect on BBM lipids or on the protein-mediated components of uptake [29, 30, 31, 38, 55]. There are several LBP in the intestine [13, 29, 30, 31, 32], but L-FABP is unlikely to play a major role, as suggested by the marked decline in jejunal L-FABP mRNA, but no alteration in lipid uptake (table 5).…”

“…Intestinal lipid uptake occurs by a process of passive permeation [29], but a component of the uptake of long-chain fatty acids is also mediated by the sodium/hydrogen exchanger and/or by lipid binding proteins in the brush border membrane or in the cytosol of the enterocyte, such as the liver fatty acid binding protein (L-FABP) and the ileal lipid binding protein (ILBP) [13, 29, 30, 31, 32]. It is unknown if the fatty acid binding proteins (FABP) in the intestine are influenced by steroids or by intestinal resection, and whether these are associated with alterations in lipid uptake.…”