Characterization of the Interaction of 2-[2-Nitro-4-(trifluoromethyl)benzoyl]-4,4,6,6-tetramethylcyclohexane-1,3,5-trione with Rat Hepatic 4-Hydroxyphenylpyruvate Dioxygenase

Publisher Website
Google Scholar
Abstract
The synthetic β-triketones are a novel family of chemicals, developed as herbicides that have activity on grass and broadleaf weeds and are selective in corn. Toxicological evaluation of a number of these chemicals has established that they interfere with rat hepatic tyrosine catabolism in vivo by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD). This paper describes the kinetics of inhibition of rat hepatic HPPD in vitro by the representative β-triketone 2-[2-nitro-4-(trifluoromethyl)benzoyl]-4,4,6,6-tetramethylcyclohexane-1,3,5-trione (1). A marked inhibition of rat hepatic HPPD was observed when 1 was incubated with the enzyme for 3 min at 37 °C prior to the initiation of the enzyme reaction by the addition of substrate. In this system, a concentration of 200 nM 1 resulted in a >90% loss of HPPD activity, and an apparent IC50 was established at approximately 50 nM. The rate constant for the inactivation of HPPD by 1 was (1.5 ± 0.2) × 10-5 s-1 nM-1 as determined by progress curve data of oxygen consumed by HPPD with time. This inhibition is reversible in that the enzyme−inhibitor complex slowly dissociates, with approximately 5.5 ± 0.6% of the enzyme activity being recovered by 6 h at 25 °C (t1/2, 25 °C, estimated at 101 ± 14 h). In short, our studies establish 1 to be a tight-binding inhibitor of rat hepatic HPPD in vitro. This inhibition is characterized by the rapid inactivation of HPPD by the formation of an enzyme−inhibitor complex that dissociates extremely slowly with recovery of enzyme activity.