title: Mutation of the sialyltransferase S-sialylmotif alters the kinetics of the donor and acceptor substrates.
abstract: Protein sequence analysis of the cloned sialyltransferase gene family has revealed the presence of two conserved protein motifs in the middle of the lumenal catalytic domain, termed L-sialylmotif and S-sialylmotif. In our previous study (Datta, A. K., and Paulson, J. C. (1995) J. Biol. Chem. 270, 1497-1500) the larger L-sialylmotif of ST6Gal I was analyzed by site-directed mutagenesis, which provided evidence that it participates in the binding of the CMP-NeuAc, a common donor substrate for all the sialyltransferases. However, none of the mutants tested in this motif had any significant effect on their binding affinities toward the acceptor substrate asialo alpha1-acid glycoprotein. In this study, we have investigated the role of the S-sialylmotif of the same enzyme ST6Gal I. In total, nine mutants have been constructed by changing the conserved amino acids of this motif to mostly alanine by site-directed mutagenesis. Kinetic analysis for the mutants which retained sialyltransferase activity showed that the mutations in the S-sialylmotif caused a change of Km values for both the donor and the acceptor substrates. Our results indicated that this motif participates in the binding of both the substrates. A sequence homology search also supported this finding, which showed that the downstream amino acid sequence of the S-sialylmotif is conserved for each subgroup of this enzyme family, indicating its association with the acceptor substrate.
authors.affiliation: Cytel Corporation and the Department of Chemistry and Molecular Biology, Scripps Research Institute, San Diego, California 92121, USA. adatta@scripps.edu
authors.forename: A K
authors.initials: AK
authors.lastname: Datta
authors.forename: A
authors.initials: A
authors.lastname: Sinha
authors.forename: J C
authors.initials: JC
authors.lastname: Paulson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D001483:Base Sequence; D019556:COS Cells; D002645:Chickens; D016384:Consensus Sequence; D006224:Cricetinae; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000378:metabolism; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D014162:Transfection; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D001212:Asialoglycoproteins; D009838:Oligodeoxyribonucleotides; D009961:Orosomucoid; D011994:Recombinant Proteins; C017030:asialoorosomucoid; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase