Spectroscopic and computational studies of NTBC bound to the non-heme iron enzyme (4-hydroxyphenyl)pyruvate dioxygenase: Active site contributions to drug inhibition

(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) is an alpha-keto-acid-dependent dioxygenase which catalyzes the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate as part of tyrosine catabolism. While several di- and tri-ketone alkaloids are known as inhibitors of HPPD and used commercially as herbicides, one such inhibitor, [2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC), has also been used therapeutically to treat type I tyrosinemia and alkaptonuria in humans. To gain further insight into the mechanism of inhibition by NTBC, a combination of CD/MCD spectroscopy and DFT calculations of HPPD/Fe-II/NTBC has been performed to evaluate the contribution of the Fe-II-NTBC bonding interaction to the high affinity of this drug for the enzyme. The results indicate that the bonding of NTBC to Fe-II is very similar to that for HPP, both involving similar pi-backbonding interactions between NTBC/HPP and Fe-II. Combined with the result that the calculated binding energy of NTBC is, in fact, similar to 3 kcal/mol less than that for HPP, the bidentate coordination of NTBC to Fell is not solely responsible for its extremely high affinity for the enzyme. Thus, the pi-stacking interactions between the aromatic rings of NTBC and two phenyalanine residues, as observed in the crystallography of the HPPD/Fe-II/NTBC complex, appear to be responsible for the observed high affinity of drug binding. (c) 2005 Published by Elsevier Inc.