Isolation and properties of car☐ylesterase P 4 from Yersinia Pseudotuberculosis

Abstract

The cardoxylesterase P 4 produced by Yersinia pseudotuberculosis was purified 330-fold by gel permeation and DEAE-trisacryl chromatography with a final yield of 21%. The apparent molecular weight, as determined by fast-protein liquid chromatography, was 45 kDa. The hydrolytic activity of esterase P 4 was higher with the 1-naphythyl esters than with the 2-naphthyl esters of acetic, propionic and butyric acids. The apparent Km values were identical for 1-naphthyl acetate and 1-naphthyl propionate (0.15 mM). The enzyme was unstable at pH values below 5, but retained 80% of its initial activity after 30 min at 65°C. It was unaffected by EDTA, eserine, tosyl-L-lysine chloromethylketone, iodoacetamide or 4-hydroxymercuribenzoate, but was strongly inhibited by low concentrations of diisopropyl fluorophosphate, suggesting the presence of serine in its active site. The purified enzyme gave a single precipitin line on Ouchterlony double immunodiffusion with homologous antiserum. This antiserum cross-reacted with the esterase bands E 3 and E 5 of Y. enterocolitica biotype 1, whereas there was no cross-reaction with the esterase bands produced by Y. enterocolitica biotypes 2 to 5, Y. intermedia, Y. frederiksenii, Y. kristensenii or Y. aldovae. The car☐ylesterase P 4 produced by Y. pestis was physicochemically, biochemically and immunologically indistinguishable from Y. pseudotuberculosis car☐ylesterase P 4. The latter enzyme and car☐ylesterase B of Escherichia coli showed some biochemical similarities, but were antigenically unrelated. Our data confirm the relevance of esterases to phylogenetic and taxonomic studies of Enterobacteria.