THE SIALYLTRANSFERASE SIALYLMOTIF PARTICIPATES IN BINDING THE DONOR SUBSTRATE CMP-NEUAC

All members of the sialyltransferase gene family cloned to date contain a conserved region, the ''sialyl-motif,'' consisting of 48-49 amino acids in the center of the coding sequence. To investigate the function of this motif, mutant constructs of the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase were designed by site directed mutagenesis, replacing 11 individual conserved amino acids with alanine. Each of the mutants was expressed in COS-1 cells, and eight of these retained sialyltransferase activity, allowing comparison of their enzymatic properties with that of the wild type enzyme. Kinetic analysis showed that six of eight mutants had a 3-12-fold higher K-m for the donor substrate CMP-NeuAc relative to the wild type enzyme, while the K-m values for the acceptor substrate were within 0.5-1.2-fold of the wild type for all eight mutants evaluated. The K-i of the donor substrate analog CDP was also evaluated for the recombinant sialyltransferase with the Val to Ala mutation at residue 220, which produced a 6-fold increase in K-m of CMP-NeuAc. A corresponding increase in K-i of 3.4-fold was observed for CDP, indicating a decreased affinity for the cytidine nucleotide. Taken together, these results suggest that the conserved sialylmotif in the sialyltransferase gene family participates in the binding of the common donor substrate, CMP-NeuAc.