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Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

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Abstract

UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man‴α1-6(GlcNAc″β1-2Man′α1-3)Manβ-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in β1-2 linkage to the 2‴-OH of the Man‴α1-6 residue. The 2‴-deoxy analogue is a competitive inhibitor (K i=0.13mm). The 2‴-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3‴-, 4‴- and 6‴-OH groups are not essential for binding or catalysis since the 3‴-, 4‴- and 6‴-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3‴-position to pentyl and substituted pentyl groups causes competitive inhibition (K i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3‴-O-(4,4-azo)pentyl group and a 3‴-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man‴α1-6 residue are essential for binding although the 2‴- and 3‴-OH face the catalytic site of the enzyme. The 4-OH group of the Manβ-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Manβ- substrate analogue. The data are compatible with our previous observations that a ‘bisecting’N-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3′-OH of the Man′α1-3 is an essential group for GlcNAc-T II since the 3′-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAcβ1-2Manα1-3Manβ-O-octyl is a good inhibitor (K i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAcβ1-2Manβ1-3Manβ- arm of the branched substrate to the enzyme is essential for catalysis.

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Authors and Affiliations

  1. Research Institute, The Hospital for Sick Children, M5G 1X8, Toronto, Ont., Canada

    Folkert Reck, Gabriele Möller, Inka Brockhausen & Harry Schachter

  2. Institut für Organische Chemie, Universität Hamburg, 20146, Hamburg, Germany

    Ernst Meinjohanns, Matthias Springer, Roland Wilkens, Johannes A. L. M. Van Dorst & Hans Paulsen

  3. Department of Biochemistry, University of Toronto, M5S 1A8, Toronto, Ont., Canada

    Inka Brockhausen & Harry Schachter

Authors
  1. Folkert Reck
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  2. Ernst Meinjohanns
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  3. Matthias Springer
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  4. Roland Wilkens
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  5. Johannes A. L. M. Van Dorst
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  6. Hans Paulsen
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  7. Gabriele Möller
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  8. Inka Brockhausen
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  9. Harry Schachter
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Additional information

Abbreviations: GlcNAc-T I, UDP-GlcNAc:Manα1-3R β(1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.

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Reck, F., Meinjohanns, E., Springer, M. et al. Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme. Glycoconjugate J 11, 210–216 (1994). https://doi.org/10.1007/BF00731220

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  • DOI: https://doi.org/10.1007/BF00731220

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