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 middl e of the lumenal catalytic domain, termed L-sialylmotif and S-sialylmo tif, 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 substrat e for all the sialyltransferases, However, none of the mutants tested in this motif had any significant effect on their binding affinities t oward the acceptor substrate asialo alpha(1)-acid glycoprotein, In thi s study, we have investigated the role of the S-sialylmotif of the sam e enzyme ST6Gal I. In total, nine mutants have been constructed by cha nging the conserved amino acids of this motif to mostly alanine by sit e-directed mutagenesis. Kinetic analysis for the mutants which retaine d sialyltransferase activity showed that the mutations in the S-sialyl motif caused a change of K-m values for both the donor and the accepto r substrates. Our results indicated that this motif participates in th e binding of both the substrates, A sequence homology search also supp orted this finding, which showed that the downstream amino acid sequen ce of the S-sialylmotif is conserved for each subgroup of this enzyme family, indicating its association with the acceptor substrate.