Purification and Characterization of a Specific 3-Deoxy- d - manno -Octulosonate 8-Phosphate Phosphatase from Escherichia coli B

Abstract

A phosphatase specific for the hydrolysis of 3-deoxy-D-manno-oculosonate (KDO)-8-phosphate was purified approximately 400-fold from crude extracts of E. coli B. The hydrolysis of KDO-8-phosphate to KDO and Pi in crude extracts of E. coli B, grown in phosphate-containing minimal medium, could be accounted for by the enzymatic activity of this specific phosphatase. No other sugar phosphate tested was an alternate substrate or inhibitor of the purified enzyme. KDO-8-phosphate phosphatase was stimulated 3- to 4-fold by the addition of 1.0 mM Co+ or Mg2+ and to a lesser extent by 1.0 mM Ba2+, Zn2+ and Mn2+. The activity was inhibited by the addition of 1.0 mM ethylenediaminetetraacetic acid, Cu2+, Ca2+, Cd2+, Hg2+ and Cl- (50% at 0.1 M). The pH optimum was determined to be 5.5-6.5 in both tris(hydroxymethyl)aminomethane-acetate and HEPES (N-2-hydroxyethylpiperazine-N''-2-ethanesulfonic acid) buffer. This specific phosphatase had an isoelectric point of 4.7-4.8 and a MW of 80,000 .+-. 6000 as determined by molecular sieving and Ferguson analysis. The enzyme appeared to be composed of 2 identical subunits of 40,000-43,000 MW. The apparent Km for KDO-8-phosphate was 5.8 .+-. 0.9 .times. 10-5 M in the presence of 1.0 mM Co2+, 9.1 .+-. 1 .times. 10-5 M in the presence of 1.0 mM Mg2+ and 1.0 .+-. 0.2 .times. 10-4 M in the absence of added Co2+ or Mg2+.