51] 3-Hexulose-6-phosphate synthase from Methylomonas (Methylococcus) capsulatus

Abstract

This chapter describes two assay methods (method I and II) and the purification procedure for 3-hexulose-6-phosphate synthase from Methylomonas (Methylococcus) capsulatus . The enzyme catalyzes a key step in the assimilation of carbon during growth of some bacteria on reduced C 1 compounds. Method 1 involves discontinuous colorimetric measurement of the rate of ribulose 5-phosphate-dependent removal of formaldehyde. Method 2 involves continuous measurement of the rate of ribulose 5-phosphate- and formaldehyde-dependent formation of hexose phosphate. Method 2 is more convenient to use but it depends on having previously purified phosphohexuloisomerase as a coupling enzyme. The purification procedure involves preparation of cell-free extract, preparation of particulate fraction, solubilization with l M NaCl, ammonium sulfate fractionation, diethylaminoethyl (DEAE)-cellulose chromatography, and calcium phosphate gel treatment. Hexulose-phosphate synthase has an absolute requirement for a bivalent metal ion—Mg 2+ and Mn 2+ being most effective and Co 2+ and Zn 2+ being much less effective. The enzyme has optimum activity, in the synthetic direction, at pH 7.0.