Mutation of the sialyltransferase S-sialylmotif alters the kinetics of the donor and acceptor substrates.
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.
Datta
A K
AK
Cytel Corporation and the Department of Chemistry and Molecular Biology, Scripps Research Institute, San Diego, California 92121, USA. adatta@scripps.edu
Sinha
A
A
Paulson
J C
JC
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
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