The inhibition of ornithine transcarbamoylase from Escherichia coli W by phaseolotoxin

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Abstract
The mechanism of inhibition of ornithine transcarbamoylase by the bacterial [Pseudomonas syringae pv. phaseolicola] toxin phaseolotoxin [cause of halo-blight in beans] [N-.delta.-(phosphosulfamyl)ornithylalanylhomoarginine] was investigated. Ornithine transcarbamoylase was purified by affinity chromatography from E. coli W argR- by using N-.delta.-(phosphonoacetyl)ornithine as the ligand. Under steady-state conditions phaseolotoxin inhibition was reversible and exhibited mixed kinetics with respect to carbamoyl phosphate. The apparent Ki and apparent K''i were 0.2 .mu.M and 10 .mu.M, respectively. Inhibition with respect to ornithine was non-competitive, with an apparent Ki of 0.9 .mu.M. These data are consistent with competitive binding of phaseolotoxin to the carbamoyl phosphate-binding site of the enzyme. The toxin also appears to be able to bind to the enzyme-carbamoyl phosphate complex, although, since K''i is 50 times greater than Ki, this event is kinetically much less significant. In the presence of phaseolotoxin ornithine transcarbamoylase exhibited a transient phase of activity before a steady state. This is consistent with low rates of association and dissociation for the toxin with enzyme and the enzyme-toxin complex. Rate constants of 2.5 .times. 104 M-1 .cntdot. s-1 and 5 .times. 10-3 s-1 were estimated for the association and dissociation constants, respectively.