Purification and some properties of the D-lactate-2-sulphatase of Pseudomonas syringae GG.
A soil bacterium grown on propan-2-yl sulphate as sole source of carbon and sulphur yielded extracts containing an enzyme capable of liberating sulphate from racemic lactate-2-sulphate. The enzyme was purified to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, batch treatment with DEAE-cellulose, and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was monomeric with an Mr of 55 000-60 000. The enzyme activity was specific for D-lactate-2-sulphate (Km 6.6 nM; maximal specific activity 14.3 mumol/min per mg of protein) and showed no activity towards the L-isomer. The products of the enzyme's action were inorganic sulphate and D-lactate which were released in equimolar amounts and stoicheiometrically with the amount of ester hydrolysed. No L-lactate was formed. Retention of configuration implied cleavage of the O-S bond of the C-O-S ester link and this was confirmed by 18O-incorporation experiments in which 18O from 18O-enriched water in the incubation medium was incorporated exclusively and quantitatively into inorganic sulphate. Only two other esters (serine-O-sulphate and p-nitrophenyl sulphate) of a total of 29 compounds tested were substrates for the enzyme. D-Lactate, L-lactate-2-sulphate and the substrate analogues glycollate-2-sulphate and butyrate-2-sulphate were significantly inhibitory.
D055598:Chemical Phenomena; D002621:Chemistry; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013429:Sulfatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D013429:Sulfatases; C043519:lactate-2-sulfate sulfatase