Kinetics of the action of thermolysin on peptide substrates

Morgan, Graham; Fruton, Joseph S.

doi:10.1021/bi00610a022

Cited by 38 publications

(36 citation statements)

References 32 publications

(34 reference statements)

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“…The turnover number of LF shows a limited variation (from one to six molecules of substrate hydrolyzed/second by one molecule of LF) upon changing the length of peptide substrates and the number of Arg residues, whose addition at the N terminus renders peptides permeable to the plasma membrane of cells (18). This figure can be compared with that of the prototype metalloproteinase, thermolysin, whose turnover is comprised in the range of 6 -16 substrate molecules hydrolyzed/second by one molecule of thermolysin (28).…”

Section: Resultsmentioning

confidence: 99%

The Metalloproteolytic Activity of the Anthrax Lethal Factor Is Substrate-inhibited

Tonello

Ascenzi

Montecucco

2003

Journal of Biological Chemistry

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The anthrax lethal factor (LF) is a Zn 2؉ endopeptidase specific for mitogen-activated protein kinase kinases (MAPKKs), which are cleaved within their N termini. Here, the proteolytic activity of LF has been investigated using novel chromogenic MAPKK-derived peptide substrates, which allowed us to determine the kinetic parameters of the reaction. LF displayed maximal proteolytic activity at the pH and temperature values of the cell cytosol, which is its site of action. LF undergoes substrate inhibition, in keeping with the non-productive binding geometry of the MAPPK-2 N terminus to LF.Toxigenic strains of Bacillus anthracis secrete three proteins: the protective antigen (PA, 1 87.2 kDa), the edema factor (EF, 88.8 kDa), and the lethal factor (LF, 90.2) (1-3). PA derives its name from the fact that immunization with it confers a protective immunity to vaccinated animals (4, 5). PA binds to a rather ubiquitous plasma membrane type I protein encoded by the tumor endothelial marker gene 8 (6, 7) and to human capillary morphogenesis protein 2 (8). PA is then proteolytically processed on the cell surface by furin and membrane furin-like peptidases, to a 63-kDa form (PA 63 ), which oligomerizes and binds LF or EF (3). The lethal toxin (PA ϩ LF) and the edema toxin (PA ϩ EF) are then endocytosed by a lipid raft-mediated clathrin-dependent process (9). Then, PA 63 undergoes an acidic triggered conformational rearrangement (10) that mediates the transfer of EF or LF from the lumen of a late endocytic compartment to the cytoplasm (11).EF is a Ca 2ϩ -and calmodulin-dependent adenylate cyclase that increases cytosolic cAMP, altering water homeostasis and the elaborate balance of intracellular signaling pathways. EF impairs neutrophil function(s), and it is believed to be responsible for the edema found in cutaneous anthrax (12).LF displays metalloproteolytic activity directed toward the N terminus of mitogen-activated protein kinase kinases (MAPKKs) (13-16). The active site zinc ion is coordinated tetrahedrally to the domain 4 of LF (residues 551-777) by a water molecule and three side chains (i.e. His-686, His-690, and Glu-735), in a structural arrangement shared by metalloproteases of the thermolysin family (17). LF has a deep and long (ϳ40 Å long) groove contiguous to the active site center, which binds peptide substrates and peptide inhibitors (17, 18). The groove has an overall negative electrostatic potential containing clusters of Glu/Asp as well as Gln/Asn residues. The determination of the sites of proteolysis of various MAPKK isoforms led to the identification of a consensus motif for the cleavage site: positively charged residues are located at positions P 7 to P 4 and hydrophobic residues are located at P 2 and P 1 Ј (Fig. 1) 2 (15). The LT metalloproteolytic activity is cytotoxic to macrophages (19), and there is evidence that it plays a major role in the development of systemic anthrax, a rapid and often fatal disease of several vertebrates including humans (20). It is worth noting that LF active site mutants ...

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Section: Resultsmentioning

confidence: 99%

The Metalloproteolytic Activity of the Anthrax Lethal Factor Is Substrate-inhibited

Tonello

Ascenzi

Montecucco

2003

Journal of Biological Chemistry

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“…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)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) with these enzymes [25, 31. 321. However,…”

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

Kunugi¹,

Koyasu²,

Kitayaki³

et al. 1996

European Journal of Biochemistry

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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,

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“…To examine more closely the validity of this suggestion, we synthesized the series Mns-(Gly),-Phe-glycinal (n = 0, 1, 2), as well as Mns-glycinal, for fluorescence studies on their interaction with papain. Mansyl [Mns, 6-(N-methylanilino)-2-naphthalene sulfonyl] derivatives of suitable substrates and inhibitors of pepsin (4), papain (5), and thermolysin (6) have been used in earlier work to study, by stopped-flow fluorescence spectroscopy, the kinetics and mechanism of the action of these enzymes. In the present investigation, the principal objective was to take advantage of the tight binding of the derivatives of Phe-glycinal at the active site of papain, as a consequence of thiohemiacetal formation by the reaction of the aldehyde group of the inhibitors with Cys-25 of the enzyme (7,8), to examine the extent of energy transfer between tryptophan in the enzyme and the mansyl group of a series of inhibitors in which the fluorescent probe group is located at various known distances from the reactive aldehyde group.…”

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“…Comparison of the inhibitor concentrations required to effect 50% inhibition indicated that the three compounds of the series Z-(Gly)n-Phe-glycinal are approximately equally effective as inhibitors of the hydrolysis of AcPhe-Gly-NA, and that they are about twice as effective in this regard as is Ac-Phe-glycinal. Examination of the members of the series Mns-(Gly)n-Phe-glycinal as inhibitors of the action of papain on 0.2 mM Ac-Phe-Gly-NA showed that the inhibitor concentration required to effect 50% inhibition at pH 6 (Fig. 1).…”

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Interaction of papain with derivatives of phenylalanylglycinal: fluorescence studies.

Henes¹,

Mattis²,

Fruton³

1979

Proc. Natl. Acad. Sci. U.S.A.

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Fluorescence studies have been performed on the interaction of papain with active-site-directed inhibitors of the type mansyl-Gly)I-Phe-glycinal, where n = 0, 1, 2. It has been found that whereas the mansyl [6(N-methylanilino-2-naphthalene sulfonyll fluorescence of mansyl-Phe-glycinal is greatly enhanced, that of the two longer mansyl compounds is not, although all three are equally effective as inhibitors of papain action. Measurements of fluorescence polarization and rotational relaxation time support the conclusion that the fluorescent probe group of the two longer mansyl compounds protrudes into the solvent to a greater degree than that of mansyl-Phe-glycinal. Considerable energy transfer from papain tryptophan to the mansyl group is evident for all three inhibitors, however, although it is most marked with mansyl-Phe-glycinal. Stopped-flow fluorescence measurements have shown that, after initial rapid interaction, the first-order conformational changes in the active-site region of papain in the complex with mansylPhe-glycinal are approximately 1/104 those observed with comparable mansyl oligopeptide substrates, and approximately 1/102 those with acetyl-Phe-glycinal.In a previous publication from this laboratory (1) it was reported that the intrinsic tryptophan fluorescence of papain is greatly enhanced upon the binding of the inhibitor Ac-Phe-glycinal (2) and of related compounds in which the acetyl group is replaced by benzyloxycarbonyl (Z), Z-Gly, or Z-Gly-Gly. This fluorescence enhancement is largely abolished by the selective oxidation of Trp-69 (3) to yield a modified papain that retains its catalytic activity toward small synthetic substrates, such as Ac-Phe-Gly-NA (NA is p-nitroaniline). The results indicated that, in the binding of the above derivatives of Phe-glycinal at the active site of papain, there is significant interaction between the phenylalanyl residue of the inhibitor and Trp-69 of the enzyme. To examine more closely the validity of this suggestion, we synthesized the series Mns-(Gly),-Phe-glycinal (n = 0, 1, 2), as well as Mns-glycinal, for fluorescence studies on their interaction with papain. Mansyl [Mns, 6-(N-methylanilino)-2-naphthalene sulfonyl] derivatives of suitable substrates and inhibitors of pepsin (4), papain (5), and thermolysin (6) have been used in earlier work to study, by stopped-flow fluorescence spectroscopy, the kinetics and mechanism of the action of these enzymes. In the present investigation, the principal objective was to take advantage of the tight binding of the derivatives of Phe-glycinal at the active site of papain, as a consequence of thiohemiacetal formation by the reaction of the aldehyde group of the inhibitors with Cys-25 of the enzyme (7,8), to examine the extent of energy transfer between tryptophan in the enzyme and the mansyl group of a series of inhibitors in which the fluorescent probe group is located at various known distances from the reactive aldehyde group. Also, the kinetics of the interaction of Mns-(Gly)n-Phe-glycinal with papain ...

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Kinetics of the action of thermolysin on peptide substrates

Cited by 38 publications

References 32 publications

The Metalloproteolytic Activity of the Anthrax Lethal Factor Is Substrate-inhibited

The Metalloproteolytic Activity of the Anthrax Lethal Factor Is Substrate-inhibited

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

Interaction of papain with derivatives of phenylalanylglycinal: fluorescence studies.

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