KD0-8-phosphate phosphatase

The reactions, 1-5 respectively, are catalyzed by D-arabinose-5-phosphate isoraerase, KD0-8-phosphate synthase, KD0-8-phosphate phosphatase, CMP-KDO synthetase and KDO transferase (s). Using E. coli B, we have isolated and purified the second, third and fourth enzymes to homogeneity and studied their properties. The fifth enzyme has been partially purified by Osborn s laboratory (15) and we have not improved on its purification or further studied its properties. 

The third enzyme in the pathway, KD0-8-phosphate phosphatase, has been purified to homogeneity (26). Because of its abosolute specificity, it should be a focal point for chemotherapeutic studies. jThe apparent for KD0-8-phosp te was+ etermined to be 5.8 x 10 M in the presence of 1.0 mM Co or Mg. This specific KD0-8-phosphate phosphatase was separated from enzymes, present in crude extracts, having phosphatase activity on other phosphorylated compounds by column chromatography on DGAE-Sephadex (26). Three distinct peaks of activity were detected. Fractions from each peak were pooled and the rates for the hydrolysis of five compounds were measured. Peak A possessed phosphatase activity for D-glucose-6-phosphate, D-arabinose-5-phosphate, D-ribose-5-phosphate and j-nitrophenylphosphate Peak B dephosphorylated D-arabinose-5-phosphate, D-ribose-5-phosphate and D-glucose-6-phos-phate. Peak C, which was well separated from the other two peaks, could only utilize KD0-8-phosphate as a substrate. KD0-8-phos-phate was not hydrolyzed by the phosphatases present in peaks A and B. 

The specificity of KD0-8-phosphate phosphatase is shown in Table IV. None of the phosphorylated sugars tested could be dephosphorylated by this enzyme, nor were they inhibitors of the reaction. All of the sugars tested (including KD0-8-phosphate) could be dephosphorylated by alkaline phosphatase. It is important to note, however, that the cells used for the isolation of KD0-8-phosphate phosphatase were grown in the presence of high inorganic phosphate which repressed the synthesis of alkaline phosphatase. It should also be noted that alkaline phosphatase is a well characterized periplasmic enzyme whereas KD0-8-phosphate... 

It was shown previously (24) that KD0-8-phosphate was a weak end-product inhibitor of the synthase reaction. Both of the end products of this reaction, KDO and inorganic phosphate are weak mixed-function inhibitors of KD0-8-phosphate phosphatase. The reduced form of KD0-8-phosphate (the C-2 carbonyl was reduced to the corresponding diasterioisomeric alcohol with NaBH, Table V, 3 red.), an open chain analogue, was neither an inhibitor of KD0-8-phosphate synthase nor was it a substrate or inhibitor of the phosphatase reaction. These findings indicate that the mechanism of KD0-8-phosphate synthase does not involve the formation of a linear intermediate and that the KD0-8-phosphate phosphatase requires the phosphorylated substrate in the ring form rather than the linear form. 

The requirement of the remaining enzymes, KD0-8-phosphate synthase, KD0-8-phosphate phosphatase and CMP-KDO synthetase, for their natural substrates, D-arabinose-5-phosphate + PEP, KD0-8-phosphate and KDO + CTP, respectively, was specific and the inhibition studies with substrate analogues were disappointing. Of the compounds tested as potential substrates of KD0-8-phosphate synthase, only the isosteric phosphonate analogue (Compound 11, Table III) of D-arabinose-5-phosphate was an alternate substrate (see Ref. 28). There were a number of weak competitive inhibitors of the synthase reaction (Compounds 2, 5, 6, 7, 15,and 19,Table III) the best inhibitor of the synthase reaction was 2-deoxy-2-fluoro-D-arabinonate-5-phosphate (compound 14, Table III). 

The requirement of the phosphatase that dephosphorylates KD0-8-phosphate is very specific (Table IV). None of the phos-phorylated sugars tested was either an inhibitor or an alternate substrate for this phosphatase. The specificity of this enzyme, the fact that none of the other intracellular phosphatases catalyze the hydrolysis of KD0-8-phosphate and the fact that KD0-8-phosphate needs to be dephosphorylated for subsequent metabolism... 

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