ENZYMOLOGICAL CHARACTERIZATION OF RECOMBINANT XENOPUS DG42, A VERTEBRATE HYALURONAN SYNTHASE

We have characterized the hyaluronan (HA) synthase activity of the Xen opus DG42 gene product in vitro, The recombinant enzyme produced in ye ast does not possess a nascent HA chain and, therefore, is an ideal mo del system for kinetic studies of the synthase's glycosyl transferase activity, The enzymatic rate was optimal from pH 7.6 to 8.1, Only the authentic sugar nucleotide precursors, UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc), were utilized to produce a l arge molecular weight polymer, UDP-glucose or the galactose epimers of the normal substrates did not substitute, The Michaelis constant, K-m , of recombinant DG42 in membranes was 60 +/- 20 and 235 +/- 40 mu M f or UDP-GlcA and UDP-GlcNAc, respectively, which is comparable to value s obtained previously from membranes derived from vertebrate cells, Th e apparent energy of activation for HA elongation is about 15 kilocalo ries/mol. DG42 polymerizes HA at average rates of about 80 to 110 mono saccharides/s in vitro, The resulting HA polysaccharide possessed mole cular weights spanning 2 x 10(6)-10(7) Da, corresponding to about 10(4 ) sugar residues, This is the first report characterizing a defined eu karyotic enzyme that can produce a glycosaminoglycan.