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 "sialylmotif," 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 Km for the donor substrate CMP-NeuAc relative to the wild type enzyme, while the Km values for the acceptor substrate were within 0.5-1.2-fold of the wild type for all eight mutants evaluated. The Ki 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 Km of CMP-NeuAc. A corresponding increase in Ki 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.
Datta
A K
AK
Cytel Corporation, San Diego, California.
Paulson
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase