Esterase activity of zinc neutral proteases

Holmquist, Barton; Vallée, Bert L.

doi:10.1021/bi00646a016

Cited by 65 publications

(31 citation statements)

References 41 publications

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“…Therefore, we endeavored to identify appropriate small molecules that have shown efficacy as enzymatic inhibitors, as a starting point for identifying candidate PME inhibitors. By analogy to mammalian tissue-degrading enzymes, we identified green tea (Camellia sinensis) catechins as potential inhibitors of PME due to their inhibition of carboxypeptidases (which also have esterase activity) during tissue remodeling in mammalian systems (Holmquist and Vallee, 1976).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of pectin methyl esterase activity by green tea catechins

Lewis¹,

Selzer

Shahar

et al. 2008

Phytochemistry

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Section: Introductionmentioning

confidence: 99%

Inhibition of pectin methyl esterase activity by green tea catechins

Lewis¹,

Selzer

Shahar

et al. 2008

Phytochemistry

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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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confidence: 99%

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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“…A widely used substrate is N-3(2-furyl)acryloylglycyl-L-leucinamide (Fagla); its hydrolysis may be followed spectrophotometrically at 345 nm (13). Thermolysin has also been shown to hydrolyze the ester analogues of suitable peptide substrates-for example, Bz-PhePla-Ala-OH (14). In specificity, therefore, thermolysin resembles pepsin and related aspartyl proteinases (3).…”

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Thermolysin-catalyzed peptide bond synthesis.

Wayne¹,

Fruton²

1983

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

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The rates of the thermolysin-catalyzed synthesis of peptides have been determined by means of HPLC. In the condensation of various N-substituted amino acids and peptides with L-leucinanilide, the enzyme exhibits preference for a hydrophobic L-amino acid as the donor of the carbonyl group of the newly formed bond. The presence of another hydrophobic amino acid residue adjacent to the carbonyl-group donor markedly enhances the rate of synthesis. In general, the effect of structural changes in both the carboxyl and amine components of the condensation reaction is in accord with the available data on the primary and secondary specificities of the thermolysin-catalyzed hydrolysis of oligopeptide substrates. A kinetic study of the condensation of benzyloxycarbonyl-L-phenylalanine with various amine components has given data on the apparent kept and Km values for the entry of the acidic component into the condensation reaction. The results are consistent with the behavior of rapid-equilibrium random bireactant systems leading to ternary enzyme-substrate complexes, with a synergistic effect in the binding of the two reactants at the active site. Because the changes in the apparent kct for the entry of the same acidic component into reaction with different amine components are greater than those in the apparent K., it is suggested that this synergism is largely expressed at the level of the transition-state complex.In a previous report from this laboratory (1), some features of the specificity of swine pepsin as a catalyst of condensation reactions leading to peptide bond synthesis were described. In reactions of the type RCOOH + NH2R' = RCO-NHR' + H20, the unfavorable equilibrium for synthesis in aqueous solution may be counteracted by the removal of the peptide product through insolubility and the use of organic co-solvents to shift the pK' of the RCOOH component (2). At suitable concentrations of the enzyme, of the appropriate reaction components, and of the co-solvent, the condensation reaction may be driven to 95-100% completion within a reasonable time period. Analysis by means of HPLC of the incubation mixture during the initial stages of the reaction permitted determination of the relative rates of peptide bond synthesis by pepsin, when the nature of RCOOH and of NH2R' was varied, and thus to determine the specificity of the enzyme as a catalyst in the condensation reaction (1). It was found that the primary specificity of pepsin with respect to the nature of the amino acid residues joined in the reaction, as well as the secondary specificity with respect to the nature of amino acids further removed from the site of peptide bond synthesis, was similar to the specificities observed previously in the study of the kinetics of the hydrolysis of oligopeptide substrates by swine pepsin (3).This approach has been applied to the proteinase thermolysin. In what follows, two aspects of the specificity of this enzyme as a catalyst of peptide bond formation are considered. First, data are presented on the initial ra...

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Esterase activity of zinc neutral proteases

Cited by 65 publications

References 41 publications

Inhibition of pectin methyl esterase activity by green tea catechins

Inhibition of pectin methyl esterase activity by green tea catechins

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

Thermolysin-catalyzed peptide bond synthesis.

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