KD0-8-phosphate synthase

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 second enzyme in the sequence studied was KD0-8-phosphate synthase. This enzyme was purified to homogeneity (24). This enzyme catalyzes the condensation of D-arabinose-5-phosphate and PEP to yield KD0-8-phosphate and inorganic phosphate. One can assay this irreversible reaction either by measuring the formation of KD0-8-phosphate or the release of P. (2Jj). The latter is the method of choice, since a number of analogues were found to interfer with the thiobarbituric acid assay. The enzyme has an apparent K for PEP of 6 x 10 M and an apparent K for D-arabinose-5-phospftiate of 2 x 10 M. m... 

TABLE III. Inhibition ot KD0-8-phosphate Synthase by Substrate Analogues... 

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 data in Table III indicate that the enzyme KD0-8-phos-phate synthase is more difficult to inhibit with phosphorylated substrate analogues than is D-arabinose-5-phosphate isomerase. Non-phosphorylated substrate analogues (Tables II) were also tested as inhibitors or substrates of KDO-8-phosphate synthase but as with D-arabinose-5-phosphate isomerase, these analogues were... 

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