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Mutation of the sialyltransferase s-sialylmotif alters the kinetics of the donor and acceptor substrates

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Overview

authors

  • Datta, A. K.
  • Sinha, A.
  • Paulson, James

publication date

  • April 1998

journal

  • Journal of Biological Chemistry  Journal

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.

subject areas

  • Amino Acid Sequence
  • Animals
  • Asialoglycoproteins
  • Base Sequence
  • COS Cells
  • Chickens
  • Consensus Sequence
  • Cricetinae
  • Cytidine Monophosphate N-Acetylneuraminic Acid
  • Humans
  • Kinetics
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligodeoxyribonucleotides
  • Orosomucoid
  • Point Mutation
  • Rats
  • Recombinant Proteins
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Sialyltransferases
  • Substrate Specificity
  • Swine
  • Transfection
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.273.16.9608

PubMed ID

  • 9545292
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Additional Document Info

start page

  • 9608

end page

  • 9614

volume

  • 273

issue

  • 16

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