3-Hexulosephosphate synthase from Methylomonas aminofaciens 77a Purification, properties and kinetics

Abstract

3-Hexulosephosphate synthase ( d -arabino-3- hexulose 6-phosphate formaldehyde lyase) was purified from an obligate methylotroph, Methylomonas aminofaciens, to homogeneity as judged by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The molecular weight was determined to be 45 000–47 000 by sedimentation velocity and gel filtration. The enzyme appears to be composed of two identical subunits ( M r = 23 000). A bivalent cation is required for the activation and stabilization of the enzyme. The enzyme is specific for formaldehyde and d-ribulose 5-phosphate. The optimum pH is 8.0 (isoelectric point, pH 5.1) and the optimum temperature is 45°C. Initial velocity studies are consistent with a sequential mechanism. The Michaelis constants are 0.29 mM for formaldehyde and 0.059 mM for d-ribulose 5-phosphate.