The inhibition of ornithine transcarbamoylase from Escherichia coli W by phaseolotoxin

Abstract

The mechanism of inhibition of ornithine transcarbamoylase by the bacterial toxin phaseolotoxin [N-δ-(phosphosulphamyl)ornithylalanylhomoarginine] was investigated. Ornithine transcarbamoylase was purified by affinity chromatography from Escherichia coli W argR- by using N-δ-(phosphonoacetyl)ornithine as the ligand. Under steady-state conditions phaseolotoxin inhibition was reversible and exhibited mixed kinetics with respect to carbamoyl phosphate. The apparent K(i) and apparent K'(i) were 0.2 μM and 10 μM respectively. Inhibition with respect to ornithine was noncompetitive, with an apparent K(i) of 0.9 μM. These data are consistent with competitive binding of phaseolotoxin to the carbamoyl phosphate-binding site of the enzyme. The toxin als appears to be able to bind to the enzyme-carbamoyl phosphate complex, although, since K'(i) is 50 times greater than K(i), 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 constant of 2.5 x 104 M-1.s-1 and 5 x 10-3s-1 were estimated for the association and dissociation constant respectively.