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Analysis of PUGNAc and NAG-thiazoline as transition state analogues for human O-GlcNAcase: mechanistic and structural insights into inhibitor selectivity and transition state poise

Academic Article
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Overview

authors

  • Whitworth, G. E.
  • Macauley, Matthew
  • Stubbs, K. A.
  • Dennis, R. J.
  • Taylor, E. J.
  • Davies, G. J.
  • Greig, I. R.
  • Vocadlo, D. J.

publication date

  • January 2007

journal

  • Journal of the American Chemical Society  Journal

abstract

  • O-GlcNAcase catalyzes the cleavage of beta-O-linked 2-acetamido-2-deoxy-beta-d-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationally modified proteins. Two potent inhibitors of this enzyme are O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2'-methyl-alpha-d-glucopyranoso[2,1-d]-Delta2'-thiazoline (NAG-thiazoline). Derivatives of these inhibitors differ in their selectivity for human O-GlcNAcase over the functionally related human lysosomal beta-hexosamindases, with PUGNAc derivatives showing modest selectivities and NAG-thiazoline derivatives showing high selectivities. The molecular basis for this difference in selectivities is addressed as is how well these inhibitors mimic the O-GlcNAcase-stabilized transition state (TS). Using a series of substrates, ground state (GS) inhibitors, and transition state mimics having analogous structural variations, we describe linear free energy relationships of log(KM/kcat) versus log(KI) for PUGNAc and NAG-thiazoline. These relationships suggest that PUGNAc is a poor transition state analogue, while NAG-thiazoline is revealed as a transition state mimic. Comparative X-ray crystallographic analyses of enzyme-inhibitor complexes reveal subtle molecular differences accounting for the differences in selectivities between these two inhibitors and illustrate key molecular interactions. Computational modeling of species along the reaction coordinate, as well as PUGNAc and NAG-thiazoline, provide insight into the features of NAG-thiazoline that resemble the transition state and reveal where PUGNAc fails to capture significant binding energy. These studies also point to late transition state poise for the O-GlcNAcase catalyzed reaction with significant nucleophilic participation and little involvement of the leaving group. The potency of NAG-thiazoline, its transition state mimicry, and its lack of traditional transition state-like design features suggest that potent rationally designed glycosidase inhibitors can be developed that exploit variation in transition state poise.

subject areas

  • Acetylglucosamine
  • Catalysis
  • Crystallography, X-Ray
  • Enzyme Activation
  • Enzyme Inhibitors
  • Glycosylation
  • Humans
  • Models, Molecular
  • Oximes
  • Phenylcarbamates
  • Substrate Specificity
  • Thermodynamics
  • Thiazoles
  • beta-N-Acetylhexosaminidases
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Identity

International Standard Serial Number (ISSN)

  • 0002-7863

Digital Object Identifier (DOI)

  • 10.1021/ja065697o

PubMed ID

  • 17227027
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Additional Document Info

start page

  • 635

end page

  • 644

volume

  • 129

issue

  • 3

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