The inactivation of ornithine transcarbamoylase by N-(N -sulpho-diaminophosphinyl)-L-ornithine

Phaseolotoxin, a tripeptide inhibitor of ornithine transcarbamoylase, is a phytotoxin produced by Pseudomonas syringae pv. phaseolicola, the causal agent of halo-blight in beans. In vivo the toxin is cleaved to release N6-(N'-sulpho-diaminophosphinyl)-Lornithine, the major toxic chemical species present in diseased leaf tissue. This paper reports on the interaction between N8-(N'-sulpho-diaminophosphinyl)-L-ornithine and ornithine transcarbamoylase. N6-(N'-Sulpho-diaminophosphinyl)-L-ornithine was found to be a potent inactivator of the enzyme, in contrast with phaseolotoxin, which previously has been reported to inhibit the enzyme reversibly. Inactivation by N6-(N'-[35S]sulpho-diaminophosphinyl)-L-ornithine resulted in the incorporation of 35S into ethanol-precipitated protein. The stoicheiometry of 35S incorporation was approximately 1 mol/mol of active sites. Inactivation was second-order and a rate constant of 106 M-1 .-1 at 0°C in 50mM-Tris/HCl, pH 9.0, was obtained. Carbamoyl phosphate, a substrate of ornithine transcarbamoylase, protected the enzyme from inactivation. A dissociation constant of 3pM for the enzyme-carbamoyl phosphate complex was calculated. L-Ornithine, the second substrate for ornithine transcarbamoylase, protected the enzyme only at high concentrations. The results are consistent with N6-(N'-sulpho-diaminophosphinyl)-L-ornithine being a potent affinity label that binds via the carbamoyl phosphate-binding site of ornithine transcarbamoylase. Cleavage of phaseolotoxin to N,-(N'-sulpho-diaminophosphinyl)L-ornithine in vivo appears to be an important function in the physiology of the disease.