Phenylalanine hydroxylase from Chromobacterium violaceum. Uncoupled oxidation of tetrahydropterin and the role of iron in hyroxylation.
A gene encoding phenylalanine hydroxylase has been cloned from Chromobacterium violaceum and expressed in Escherichia coli. The purified phenylalanine hydroxylase contains copper, which does not support enzymatic activity. Upon removal of copper by dithiothreitol (DTT), the enzyme contains substoichiometric amounts of calcium and zinc but little or no redox-active metal ions. The copper-depleted hydroxylase catalyzes the phenylalanine-dependent oxidation of 6, 7-dimethyltetrahydropterin (DMPH4) by O2 in a reaction in which phenylalanine is not hydroxylated and does not appear to undergo a chemical change, and hydrogen peroxide is produced. Analogs of phenylalanine also activate the oxidation of DMPH4. Both the copper-phenylalanine hydroxylase and the copper-depleted hydroxylase catalyze the hydroxylation of phenylalanine in the presence of DTT and FeSO4 in a reaction in which hydrogen peroxide is not produced. The apparent values of Km for Fe2+ and DTT are 0.28 microM and 1.1 mM, respectively, at 1.0 mM phenylalanine, 120 microM DMPH4 and pH 7. 4 and 23 degreesC. The apparent value of kcat is 14.3 s-1 under these conditions. Glutathione, mercaptoethanol, and dihydrolipoate support the hydroxylation of phenylalanine essentially as well as DTT. Incubation of copper-depleted hydroxylase with FeSO4, phenylalanine, and DTT followed by gel permeation chromatography leads to an iron-hydroxylase containing approximately 1 molecule of iron per molecule of enzyme. The iron-hydroxylase displays an optical absorption band extending from 300 to 600 nm, and it catalyzes the hydroxylation of phenylalanine at the same maximum rate as the iron-activated hydroxylase but does not require added Fe2+. We conclude that iron participates in the hydroxylation of phenylalanine. Iron is not required for the oxidation of DMPH4, although it may exert a modest acceleration effect. A hypothetical mechanism is presented wherein the reaction of iron with the putative 4a-hydroperoxy-DMPH4 leads to 4a-hydroxy-DMPH4 and a high valent iron-oxy species. The iron-oxy species is postulated to react with phenylalanine in the hydroxylation process.
Chen
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Institute for Enzyme Research, The Graduate School and Department of Biochemistry, College of Agricultural and Biological Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.
Frey
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PA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D002861:Chromobacterium / Q000201:enzymology*; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006861:Hydrogen Peroxide / Q000378:metabolism; D006900:Hydroxylation; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry; D008670:Metals / Q000032:analysis; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010100:Oxygen / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000737:chemistry*; D011621:Pteridines / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017421:Sequence Analysis; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D001426:Bacterial Proteins; D008667:Metalloproteins; D008670:Metals; D011621:Pteridines; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D010649:Phenylalanine; C005063:2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine; D003300:Copper; D006861:Hydrogen Peroxide; D007501:Iron; D010651:Phenylalanine Hydroxylase; D010100:Oxygen