Synthesis and inhibition properties of a series of pyranose derivatives towards a Zn-metalloproteinase from Saccharomonospora canescens.

The Zn-proteinase, isolated from Saccharomonospora canescens (NPS), shares many common features with thermolysin, but considerable differences are also evident, as far as the substrate recognition site is concerned. In substrates of general structure AcylAlaAlaPhe 4NA, this neutral proteinase cleaves only the arylamide bond (non-typical activity of Zn-proteinases), while thermolysin attacks the peptide bond Ala-Phe. Phosphoramidon is a powerful tight binding inhibitor for thermolysin and significantly less specific towards NPS.

Do N-terminal nucleophile hydrolases indeed have a single amino acid catalytic center?

A new set of experimental kinetic data on the hydrolysis of a series of phenylacetyl p-substituted anilides catalyzed by penicillin G acylase from Escherichia coli (PGA) is presented in this article. The Hammett plot of log(k(cat,R)/k(cat,H)) versus sigma(p) (-) has three linear segments, which distinguishes the enzyme from the other N-terminal nucleophile hydrolases for which data are available. Three amino acids in the vicinity of the catalytic SerB1 (AsnB241, AlaB69, and GlnB23) were included in the quantum mechanical model.

Penicillin amidase-catalysed transfer of low specific acyl moiety. Synthesis of 7-benzoxazolonylacetamido desacetoxycephalosporanic acid.

The methyl ester of 2-benzoxazolon-3-yl-acetic acid was used as an acyl donor in the penicillin amidase-catalysed transfer reaction to 7-aminodesacetoxycephalosporanic acid. The synthesis of 7-(2-benzoxazolon-3-yl-acetamido)-desacetoxycephalosporanic acid was carried out as a kinetically controlled reaction. A characteristic feature of this system is that the benzoxazolone derivatives are very low specific substrates for penicillin amidase (the kcat/Km values for their hydrolysis were shown to be 10(5)-fold lower compared to the corresponding values for phenylacetyl derivatives).

N-anthraniloyl-Ala-Ala-Phe-4-nitroanilide, a highly sensitive substrate for subtilisins.

A new substrate for subtilisins, anthraniloyl-Ala-Ala-Phe-4-nitroanilide, has been synthesized and characterized. The peptide is a fluorogenic substrate that is intramolecularly quenched without loss of its chromogenic properties and offers a possibility for double-assay kinetic analysis. The kinetic parameters determined for subtilisin Carlsberg are Km = 0.

Secondary interactions in mesentericopeptidase-catalyzed hydrolysis of peptide ester and 4-nitroanilide substrates.

Five substrate series with the formulae Z-(Gly)n-Phe-OMe, Z-(Ala)n-Phe-OMe, Ac-(Ala)n-Phe-OMe, Z-(Gly)n-Phe-NA, and Suc-(Gly)n-Phe-NA (n = 0-2) (Z-benzyloxycarbonyl) were synthesized and used to study the active site of mesentericopeptidase (EC 3.4.21).

Kinetic study of the effect of heparin on the amidase activity of trypsin, plasmin and urokinase.

It was found that the effect of heparin on the amidase activity of urokinase (E C 3.4.21.

A study of the alkaline mesentericopeptidase active site by means of peptide chloromethyl ketones.

The kinetics of inactivation of alkaline mesentericopeptidase was studied using chloromethyl ketone derivatives of amino acids and peptides. It was shown that Tos-LysCH2Cl and Tos-PheCH2Cl did not influence the enzyme activity, while the inhibitory effect of Cbz-Ala-Gly-PheCH2Cl was 35 times that of Cbz-Ala-PheCH2Cl. The dependence of the pseudo-first order rate constant of the enzyme inactivation by Cbz-Ala-Gly-PheCH2Cl on pH and temperature indicated that a group with a pK of 6.

Primary specificity of alakaline mesentericopeptidase. Kinetic parameters for the hydrolysis of alpha-N-acetyl-L-amino acid methyl esters.

The steady-state kinetics of the alkaline mesentericopeptidase-catalysed hydrolysis of esters of the general formula Ac-X-OMe(OEt) has been studied, "X" being an amino acid residue (Ala, Val, Leu, Ile, Phe, Tyr, Trp, Lys). The values of the specificity ratio kcat/Km indicate that the bonds involving the carboxyl group of amino acids with aromatic and bulky aliphatic side chain are hydrolysed most effectively. On account of this, alkaline mesentericopeptidase is classified as a chymotrypisn-like alkaline protease.

Structure-activity relationship in the urokinase hydrolysis of alpha-N-acetyl-L-lysine anilides.

The absence of both nonproductive binding and substrate activation and also the good solubility of the substrates make the urokinase-catalysed hydrolysis of specific anilides a very suitable reaction for substrate structure-enzyme activity studies. Derivatives of alpha-N-acetyl-L-lysine anilide with high sigma minus-value substituents in the aniline ring were synthesized. Rate constants kappa-cat.