Catalytic, NonCatalytic, and Inhibitory Phenomena: Kinetic Analysis of (4-Hydroxyphenyl)pyruvate Dioxygenase from Arabidopsis thaliana

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Abstract

(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) incorporates both atoms of molecular oxygen into 4-hydroxyphenylpyruvate (HPP) to form homogentisate (HG). This reaction has direct relevance in both medicine and agriculture. In humans, the specific inhibition of HPPD alleviates the symptoms of diseases that arise from tyrosine catabolism defects. However, in plants, the inhibition of HPPD bleaches, stunts, and ultimately kills the organism. The reason for this is that in mammalian metabolism the product HG does not feed into other pathways, whereas in plants it is the precursor for the redox active portion of tocopherols and plastoquinones. There are a number of commercially available herbicides that directly target the inhibition of the HPPD reaction. Plant HPPD however is largely uncharacterized in terms of its catalysis and inhibition reactions. In this study, we examine the catalysis and inhibition of HPPD from Arabidopsis thaliana (AtHPPD). We have expressed AtHPPD and purified the enzyme to high specific activity. This form of HPPD accumulates two transient species in single turnover reactions with the native substrate HPP. These transients appear to be equivalent to intermediates I and III observed in the enzyme from Streptomyces (Johnson-Winters et al. (2005), Biochemistry, 44, 7189-7199). The first intermediate is a relatively strongly absorbing species with maxima at 380 and 490 nm. This species decays to a second intermediate that is fluorescent and has been assigned as the complex of the enzyme with the product, HG. The decay of this intermediate is rate-determining in multiple turnover reactions. The reaction of the enzyme with the analogue of the substrate, phenylpyruvate (PPA), is noncatalytic. A single turnover reaction is observed with this ligand that renders the enzyme oxidized to the ferric form, consumes a stoichiometric amount of dioxygen, and yields 66% phenylacetate as a product. Additional absorbance features at 365 and 670 nm accumulate during inactivation and give the inactivated enzyme a green color but has the same molecular mass as the active enzyme as determined by mass spectrometry.

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