Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli

Abstract

3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase was purified 450-fold from frozen E. coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+ and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to be 45.degree. C, and the energy of activation calculated by the Arrhenius equation was 15,000 calories (approximately 3585 J)/mol. The enzyme activity was pH and buffer dependent, showing 2 pH optima, 1 at pH 4.0-6.0 in succinate buffer and 1 at pH 9.0 in glycine buffer. The isoelectric point of the enzyme was 5.1. KDO-8-phosphate synthetase had a MW of 90,000 .+-. 6000 as determined by molecular sieving through Sephadex G-200 and by Ferguson analysis using polyacrylamide gels. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 90,000 MW native enzyme was composed of 3 identical subunits, each with an apparent MW of 32,000 .+-. 4000. The enzyme had an apparent Km for D-arabinose-5-phosphate of 2 .times. 10-5 M and an apparent Km for PEP of 6 .times. 10-6 M. No other sugar or sugar-phosphate could substitute for D-arabinose-5-phosphate. D-Ribose-5-phosphate was a competitive inhibitor of D-arabinose-5-phosphate, with an apparent Ki [inhibition constant] of 1 .times. 10-3 M. The purified enzyme was utilized to synthesize millimole quantities of pure KDO-8-phosphate.