Mechanism of inactivation of ornithine transcarbamoylase by Ndelta -(N'-Sulfodiaminophosphinyl)-L-ornithine, a true transition state analogue? Crystal structure and implications for catalytic mechanism.
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.
Langley
D B
DB
Department of Biochemistry, The University of Sydney, Sydney 2006, Australia and the Horticultural and Food Research Institute of New Zealand, Mt Albert Research Centre, Auckland 1003, New Zealand.
Templeton
M D
MD
Fields
B A
BA
Mitchell
R E
RE
Collyer
C A
CA
D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D009952:Ornithine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D009954:Ornithine Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013997:Time Factors
D008024:Ligands; C046037:octicidine; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase; D009584:Nitrogen