Observation of covalent intermediates in an enzyme mechanism at atomic resolution

Science. 2001 Oct 12;294(5541):369-74. doi: 10.1126/science.1063601.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

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


  • Ligands
  • Protons
  • Ribosemonophosphates
  • Schiff Bases
  • Water
  • 2-deoxyribose 5-phosphate
  • Aldehyde-Lyases
  • deoxyribose-phosphate aldolase
  • Lysine

Associated data

  • PDB/1JCJ
  • PDB/1JCL