Inhibition of p-hydroxyphenylpyruvate dioxygenase by the diketonitrile of isoxaflutole: A case of half-site reactivity

p-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homog entisate from p-hydroxyphenylpyruvate and molecular oxygen. In plants, this enzyme is the molecular target of new families of very active bleaching he rbicides. In the study presented here, we report for the first time on the purification to homogeneity of a plant enzyme, as obtained from recombinant Escherichia coli cells expressing a cDNA encoding carrot HPPD. The purifie d enzyme allowed us to carry out a detailed characterization of the inhibit ory properties of a diketonitile (DKN), the active inhibitor formed from th e benzoylisoxazole herbicide isoxaflutole. Inhibition kinetic analyses conf irmed that DKN exerts a slow and tight-binding inhibition of HPPD, competit ive with respect to the p-hydroxyphenylpyruvate substrate. The stoichiometr y of DKN binding to HPPD determined by kinetic analyses or by direct bindin g of [C-14]- DKN revealed a half-site reactivity of DKN.