Abstract
Purified preparations of 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. strain P. J. 874 are blue, epsilon 595-850 approximately 2.6 +/- 0.5 (n = 6) mM-1 cm-1. Iron and zinc were the only metals detected by x-ray fluorescence of an enzyme preparation and the mean content in different preparation as determined by atomic absorption spectroscopy was determined by atomic absorption spectroscopy was 0.95 +/- 0.17 (n = 6) and 0.68 +/- 0.27 (n = 7) mol/mol 150-kilodalton tetramer, respectively. It is yet unclear if zinc is a contaminant or may be given a structural role. Results with iron chelators and reductants showed that the 595-nm absorbance is linked to enzyme-bound Fe3+ and that reduction of iron, which occurs concomitantly with disappearance of the color, is required for enzyme activity. The enol tautomer of 4-hydroxyphenylpyruvate appeared to form 2:1 a complex with enzyme-bound Fe3+, which may be the cause of the long known substrate inhibition of the enzyme. Iron chelation also seemed to be involved in the inhibition by other substrate analogues, i.e. substituted catechols and those with one phenolic hydroxyl group in ortho position to short carboxylic acid side chains. Together, substrate analogue, pH, and modification studies indicated that the tautomerizable keto group with a double bond in 3-4 position favors productive substrate binding to Fe2+ and a base with a pK alpha of approximately 6.4.
Highlights
- dioxygenase from Pseudomonas sp. strain P
Two 2-oxoacid-dependent dioxygenases, 4-hydroxyphenylpyruvate dioxygenase[12], and thymine 7-hydroxylase [13] have an ordered sequential mechanism, winhich O2binds as the last substrate and C 0 2leaves as the fist product
This order of substrate binding is the same as that found for most oxygenases e.g. tryptophan 2,3-dioxygenase [14], protocatechuate 3,4-dioxygenase [15], and P-450 cytochromes [16, 17]
Summary
Two 2-oxoacid-dependent dioxygenases, 4-hydroxyphenylpyruvate dioxygenase[12], and thymine 7-hydroxylase [13] have an ordered sequential mechanism, winhich O2binds as the last substrate and C 0 2leaves as the fist product. The pseudomonad pendent dioxygenases [26] involved a primary attack by oxyenzyme was a 150-kilodaltontetramer of apparently identical gen at the position to be hydroxylated. These mechanisms subunits [2]. Experimental support for sucahmechanism or is anobligatory requirement as is the case with the present is given by the uncoupling, z.e. formation of COn without enzyme [2] The enzymes of this type are all stimulated by hydroxylation,whichoccurswith 2-oxoglutarate-dependent catalase.
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