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On the role of the conserved aspartate in the hydrolysis of the phosphocysteine intermediate of the low molecular weight tyrosine phosphatase

Academic Article
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Overview

authors

  • Asthagiri, D.
  • Liu, T. Q.
  • Noodleman, Louis
  • Van Etten, R. L.
  • Bashfordt, D.

publication date

  • October 2004

journal

  • Journal of the American Chemical Society  Journal

abstract

  • The usual rate-determining step in the catalytic mechanism of the low molecular weight tyrosine phosphatases involves the hydrolysis of a phosphocysteine intermediate. To explain this hydrolysis, general base-catalyzed attack of water by the anion of a conserved aspartic acid has sometimes been invoked. However, experimental measurements of solvent deuterium kinetic isotope effects for this enzyme do not reveal a rate-limiting proton transfer accompanying dephosphorylation. Moreover, base activation of water is difficult to reconcile with the known gas-phase proton affinities and solution phase pK(a)'s of aspartic acid and water. Alternatively, hydrolysis could proceed by a direct nucleophilic attack by a water molecule. To understand the hydrolysis mechanism, we have used high-level density functional methods of quantum chemistry combined with continuum electrostatics models of the protein and the solvent. Our calculations do not support a catalytic activation of water by the aspartate. Instead, they indicate that the water oxygen directly attacks the phosphorus, with the aspartate residue acting as a H-bond acceptor. In the transition state, the water protons are still bound to the oxygen. Beyond the transition state, the barrier to proton transfer to the base is greatly diminished; the aspartate can abstract a proton only after the transition state, a result consistent with experimental solvent isotope effects for this enzyme and with established precedents for phosphomonoester hydrolysis.

subject areas

  • Animals
  • Aspartic Acid
  • Cattle
  • Cysteine
  • Hydrolysis
  • Kinetics
  • Models, Molecular
  • Molecular Weight
  • Phosphorylation
  • Protein Tyrosine Phosphatases
  • Quantum Theory
  • Thermodynamics
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Identity

International Standard Serial Number (ISSN)

  • 0002-7863

Digital Object Identifier (DOI)

  • 10.1021/ja048638o

PubMed ID

  • 15453802
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Additional Document Info

start page

  • 12677

end page

  • 12684

volume

  • 126

issue

  • 39

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