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Observation of covalent intermediates in an enzyme mechanism at atomic resolution

Academic Article
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Overview

related to degree

  • Mitchell, Michael, Ph.D. in Chemistry, Scripps Research 1997 - 2002

authors

  • Heine, A.
  • DeSantis, G.
  • Luz, J. G.
  • Mitchell, Michael
  • Wong, Chi-Huey
  • Wilson, Ian

publication date

  • October 2001

journal

  • Science  Journal

abstract

  • In classical enzymology, intermediates and transition states in a catalytic mechanism are usually inferred from a series of biochemical experiments. Here, we derive an enzyme mechanism from true atomic-resolution x-ray structures of reaction intermediates. Two ultra-high resolution structures of wild-type and mutant d-2-deoxyribose-5-phosphate (DRP) aldolase complexes with DRP at 1.05 and 1.10 angstroms unambiguously identify the postulated covalent carbinolamine and Schiff base intermediates in the aldolase mechanism. In combination with site-directed mutagenesis and (1)H nuclear magnetic resonance, we can now propose how the heretofore elusive C-2 proton abstraction step and the overall stereochemical course are accomplished. A proton relay system appears to activate a conserved active-site water that functions as the critical mediator for proton transfer.

subject areas

  • Aldehyde-Lyases
  • Amino Acid Substitution
  • Binding Sites
  • Catalysis
  • Chemistry, Physical
  • Crystallization
  • Crystallography, X-Ray
  • Escherichia coli
  • Hydrogen Bonding
  • Hydrogen-Ion Concentration
  • Ligands
  • Lysine
  • Models, Chemical
  • Mutagenesis, Site-Directed
  • Mutation
  • Nuclear Magnetic Resonance, Biomolecular
  • Physicochemical Phenomena
  • Protein Conformation
  • Protein Folding
  • Protein Structure, Tertiary
  • Protons
  • Ribosemonophosphates
  • Schiff Bases
  • Water
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Identity

International Standard Serial Number (ISSN)

  • 0036-8075

Digital Object Identifier (DOI)

  • 10.1126/science.1063601

PubMed ID

  • 11598300
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Additional Document Info

start page

  • 369

end page

  • 374

volume

  • 294

issue

  • 5541

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