The administration of the compound 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC) to rats (10 mg/kg body wt) caused an elevation in the concentration of plasma
tyrosine and gave products in urine that were identified as
4-hydroxyphenylpyruvate (HPPA) and 4-hydroxyphenyllactate (
HPLA). This observed chemically induced
tyrosinemia established that this compound perturbs
tyrosine catabolism and suggested that the causal effect is the inhibition of
4-hydroxyphenylpyruvate dioxygenase (HPPD). This was confirmed when rat liver HPPD was found to be markedly inhibited by NTBC when the
enzyme and chemical were incubated, in vitro, for 3 min at 37 degrees C prior to the initiation of the
enzyme reaction by the addition of substrate. At 100 nM NTBC, approximately 90% of the
enzyme activity was lost and an IC50 was calculated at approximately 40 nM. The inhibition of HPPD by NTBC (50 nM) is time-dependent; the
enzyme activity was reduced by > 50% within 30 sec. Progress curve data of loss of
enzyme activity with time gave a rate constant for the inactivation of rat liver HPPD [k*, formation of an HPPD-inhibitor (EI) complex] by NTBC of 9.9 +/- 2.5 x 10(-5) sec-1 nM-1. It was established that NTBC is not irreversibly bound in the EI complex but slowly dissociates with a recovery of
enzyme activity of 13.7 +/- 1.0% over a 7-hr period (t1/2, 25 degrees C estimated at 63 hours). In comparison, the compound 2-(2-chloro-4-methanesulfonylbenzoyl)-cyclohexane-1,3-dione (CMBC), an analog of NTBC, gave a similar rate for the inactivation of HPPD (k*, 3.3 +/- 0.8 x 10(-5) sec-1 nM-1), whereas 45 +/- 8% of the
enzyme activity was recovered over a 7-hr period (t1/2, 25 degrees C approximately 10 hr). These studies establish that NTBC and CMBC are potent, time-dependent (tight-binding) reversible inhibitors of HPPD. The inhibition is characterized by a rapid inactivation of the
enzyme by the formation of an HPPD-inhibitor complex that dissociates with recovery of
enzyme activity. In vivo, the inhibition of HPPD causes a
tyrosinemia that abates with the recovery of
enzyme activity. The understanding of the mechanism by which NTBC perturbs
tyrosine catabolism has led to the clinical use of this chemical as the first effective pharmacological
therapy for the hereditary disorder
tyrosinemia I.