CHARACTERIZATION OF THE INTERACTION OF ZOYL]-4,4,6,6-TETRAMETHYL-CYCLOHEXANE-1,3,5-TRIONE WITH RAT HEPATIC 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE

The synthetic beta-triketones are a novel family of chemicals, develop ed as herbicides that have activity on grass and broadleaf weeds and a re selective in corn. Toxicological evaluation of a number of these ch emicals has established that they interfere with rat hepatic tyrosine catabolism in vivo by inhibiting the enzyme 4-hydroxyphenylpyruvate di oxygenase (HPPD). This paper describes the kinetics of inhibition of r at hepatic HPPD in vitro by the representative beta-triketone nzoyl]-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 fo r 3 min at 37 degrees 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 wa s established at approximately 50 nM. The rate constant for the inacti vation of HPPD by 1 was (1.5 +/- 0.2) x 10(-5) s(-1) nM(-1) as determi ned by progress curve data of oxygen consumed by HPPD with time. This inhibition is reversible in that the enzyme-inhibitor complex slowly d issociates, with approximately 5.5 +/- 0.6% of the enzyme activity bei ng recovered by 6 h at 25 degrees C (t(1/2), 25 degrees C, estimated a t 101 +/- 14 h). In short, our studies establish 1 to be a tight-bindi ng inhibitor of rat hepatic HPPD in vitro. This inhibition is characte rized by the rapid inactivation of HPPD by the formation of an enzyme- inhibitor complex that dissociates extremely slowly with recovery of e nzyme activity.