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

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Abstract
Phaseolotoxin, a tripeptide inhibitor of ornithine transcarbamoylase OTC, 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 N.delta.-(N''-sulfo-diaminophosphinyl)-L-ornithine (Psorn), the major toxic chemical species present in diseased leaf tissue. The interaction between Psorn and OTC was investigated. Psorn was a potent inactivator of the enzyme, in contrast with phaseolotoxin, which previously has been reported to inhibit the enzyme reversibly. Inactivation by [35S]Psorn resulted in the incorporation of 35S into ethanol-precipitated protein. The stoicheiometry of 35S incorporation was .apprx. 1 mol/mol of active sites. Inactivation was 2nd-order and a rate constant of 106 M-1 .cntdot. s-1 at 0.degree. C in 50 mM-Tris/HCl, pH 9.0, was obtained. Carbamoyl phosphate, a substrate of OTC, protected the enzyme from inactivation. A Kd of 3 .mu.M for the enzyme-carbamoyl phosphate complex was calculated. L-Ornithine, the 2nd substrate for OTC, protected the enzyme only at high concentrations. The results are consistent with Psorn being a potent affinity label that binds via the carbamoyl phosphate-binding site of OTC. Cleavage of phaseolotoxin to Psorn in vivo appears to be an important fucntion in the physiology of the disease.