Mechanism of inactivation of ornithine transcarbamoylase by Ndelta -(N'-Sulfodiaminophosphinyl)-L-ornithine, a true transition state analogue? Crystal structure and implications for catalytic mechanism

J Biol Chem. 2000 Jun 30;275(26):20012-9. doi: 10.1074/jbc.M000585200.

Abstract

The crystal structure is reported at 1.8 A resolution of Escherichia coli ornithine transcarbamoylase in complex with the active derivative of phaseolotoxin from Pseudomonas syringae pv. phaseolicola, N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine. Electron density reveals that the complex is not a covalent adduct as previously thought. Kinetic data confirm that N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine exhibits reversible inhibition with a half-life in the order of approximately 22 h and a dissociation constant of K(D) = 1.6 x 10(-12) m at 37 degrees C and pH 8.0. Observed hydrogen bonding about the chiral tetrahedral phosphorus of the inhibitor is consistent only with the presence of the R enantiomer. A strong interaction is also observed between Arg(57) Nepsilon and the P-N-S bridging nitrogen indicating that imino tautomers of N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine are present in the bound state. An imino tautomer of N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine is structurally analogous to the proposed reaction transition state. Hence, we propose that N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine, with its three unique N-P bonds, represents a true transition state analogue for ornithine transcarbamoylases, consistent with the tight binding kinetics observed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Catalysis
  • Crystallography, X-Ray
  • Electrons
  • Escherichia coli / enzymology
  • Kinetics
  • Ligands
  • Models, Molecular
  • Molecular Sequence Data
  • Nitrogen / metabolism
  • Ornithine / analogs & derivatives*
  • Ornithine / chemistry
  • Ornithine / pharmacology
  • Ornithine Carbamoyltransferase / antagonists & inhibitors
  • Ornithine Carbamoyltransferase / chemistry
  • Ornithine Carbamoyltransferase / metabolism*
  • Protein Conformation
  • Time Factors

Substances

  • Ligands
  • octicidine
  • Ornithine
  • Ornithine Carbamoyltransferase
  • Nitrogen

Associated data

  • PDB/1DUV