Isolation and properties of carboxylesterase P4 from Yersinia pseudotuberculosis.
The carboxylesterase P4 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 P4 was higher with the 1-naphthyl 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 degrees 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 E3 and E5 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 carboxylesterase P4 produced by Y. pestis was physicochemically, biochemically and immunologically indistinguishable from Y. pseudotuberculosis carboxylesterase P4. The latter enzyme and carboxylesterase 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.
Goullet
P
P
Laboratoire de Microbiologie, Faculté X. Bichat, Université Paris VII.
Branger
C
C
Philippe
J C
JC
Picard
B
B
D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D003429:Cross Reactions; D004926:Escherichia coli / Q000201:enzymology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D013045:Species Specificity; D013379:Substrate Specificity; D015007:Yersinia / Q000201:enzymology; D015011:Yersinia pseudotuberculosis / Q000145:classification / Q000201:enzymology*
D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase