Mk. Ellis et al., CHARACTERIZATION OF THE INTERACTION OF ZOYL]-4,4,6,6-TETRAMETHYL-CYCLOHEXANE-1,3,5-TRIONE WITH RAT HEPATIC 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE, Chemical research in toxicology, 9(1), 1996, pp. 24-27
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.