Pterin-4a-carbinolamine dehydratase from Pseudomonas aeruginosa: characterization, catalytic mechanism and comparison to the human enzyme.

The three-dimensional structure of pterin-4a-carbinolamine dehydratase (PCD) from Pseudomonas aeruginosa has been solved. Based on this we have investigated the roles of putative active center residues through functional replacement by site-directed mutagenesis. Three histidines, His73, His74 and His91, appear to be involved in dehydration catalysis. The three-dimensional positions of these residues match those of corresponding histidines at the active center of human PCD. Based on the coincidence of catalytic parameters, and on the similar effects induced by the mutations, it is concluded that the substrate binding mode and the reaction mechanisms of bacterial and human PCD are basically identical.

Location of the active site and proposed catalytic mechanism of pterin-4a-carbinolamine dehydratase.

Based on the recently solved three-dimensional structure of pterin-4a-carbinolamine dehydratase from rat/human liver the involvement of the proposed active-site residues Glu57, Asp60, His61, His62, Tyr69, His79, Arg87 and Asp88 was examined by site-directed mutagenesis. Most of the mutants showed reduced activity, and only the Glu57-->Ala mutant and the His61-->Ala, His62-->Ala double mutant were fully devoid of activity. The dissociation constants of quinonoid 6,6-dimethyl-7,8-dihydropterin were significantly increased for binding to the Glu57-->Ala, His61-->Ala, His62-->Ala single mutants and the His61-->Ala, His62-->Ala double mutant, confirming that His61 and His62 are essential for substrate binding and catalysis. The mechanism of dehydration is proposed to involve base catalysis at the N(5)-H group of the substrate by His61.

Human pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1 alpha. Characterization and kinetic analysis of wild-type and mutant enzymes.

Pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor-1 alpha is a protein with two different functions. We have overexpressed and purified the human wild-type protein, and its Cys81Ser and Cys81Arg mutants. The Cys81Arg mutant has been proposed to be causative in a hyperphenylalaninaemic patient [Citron, B. A., Kaufman, S., Milstien, S., Naylor, E. W., Greene, C. L. & Davis, M. D. (1993) Am. J. Hum. Genet. 53, 768-774]. The dehydratase behaves as a tetramer on gel filtration, while cross-linking experiments showed mono-, di-, tri-, and tetrameric forms, irrespective of the presence of the single Cys81. Sulfhydryl-modifying reagents did not affect the activity, but rather showed that Cys81 is exposed. Various pterins bind and quench the tryptophan fluorescence suggesting the presence of a specific binding site. The fluorescence is destroyed upon light irradiation. Wild-type and the Cys81Ser protein enhance the rate of the phenylalanine hydroxylase assay approximately 10-fold, a value similar to that of native dehydratase from rat liver; the Cys81Arg mutant, in contrast, has significantly lower activity. This is compatible with the hypothesis that the dehydratase is a rate-limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spontaneous dehydration of the synthetic substrate 6,6-dimethyl-7,8-dihydropterin-4a-carbinolamine approximately 50-70-fold at 4 degrees C and pH 8.5. The results are discussed in view of the recently solved three-dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier, G. & Suck, D. (1995) EMBO J. 14, 2032-2042].

Effect of high-protein meal plus aspartame ingestion on plasma phenylalanine concentrations in obligate heterozygotes for phenylketonuria.

The effect of a protein-rich meal alone or in combination with 85 mumol/kg body weight aspartame (APM) on plasma phenylalanine and large neutral amino acids (LNAA) was evaluated in obligate heterozygotes for phenylketonuria (PKU) and normal subjects (controls). Thirteen PKU heterozygotes (seven women, six men) and 13 controls (five women, eight men) ingested a 12-noon meal providing approximately 303 mumol/kg Phe. In addition, 10 PKU heterozygotes (five women, five men) and 10 controls (five women, five men) ingested the same meal with 85 mumol/kg APM (providing 75 mumol/kg Phe). Plasma amino acids were analyzed at baseline (-4 and 0 hours) and at 1, 3, and 20 hours after the meal or meal plus APM. Compared with the meal alone, ingestion of the meal plus APM significantly increased plasma Phe concentrations in both controls and PKU heterozygotes. Mean plasma Phe values were higher for controls at 1 hour (95 +/- 7 mumol/L) and for PKU heterozygotes at 3 hours (153 +/- 21 mumol/L). After the addition of APM to the meal, the highest mean plasma Phe concentration was only slightly greater than the usual postprandial range for both controls and PKU heterozygotes. Ingestion of the meal did not increase the plasma Phe/LNAA ratio in either controls or PKU heterozygotes. Compared with baseline, the plasma Phe/LNAA ratio increased significantly 1 hour after combined ingestion of the meal plus APM in both groups (P = .020 and P = .008, respectively); however, the ratios were well below the range of Phe/LNAA values in individuals with mild hyperphenylalaninemia, who are clinically normal and do not require a Phe-restricted diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenylalanine hydroxylase-stimulating protein/pterin-4 alpha-carbinolamine dehydratase from rat and human liver. Purification, characterization, and complete amino acid sequence.

Phenylalanine hydroxylase-stimulating protein, also known as pterin-4 alpha-carbinolamine dehydratase (PHS/PCD), was purified from rat and, for the first time, from human liver. We obtained their complete protein primary sequence using a combination of liquid secondary ionization mass spectrometry/tandem quadrupole mass spectrometry, electrospray ionization mass spectrometry, and Edman microsequence analysis. The amino acid sequences of human and rat PHS/PCD were found to be identical. Surprisingly, the primary structure of PHS/PCD is also essentially identical to a protein of the cell nucleus, named dimerization cofactor of hepatocyte nuclear factor 1 alpha, recently reported to be involved in transcription (Mendel, D. M., Khavari, P. A., Conley, P. B., Graves, M. K., Hansen, L. P., Admon, A., and Crabtree, G. R. (1991) Science 254, 1762-1767).

7-substituted pterins in humans with suspected pterin-4a-carbinolamine dehydratase deficiency. Mechanism of formation via non-enzymatic transformation from 6-substituted pterins.

A recently described new form of hyperphenylalaninemia is characterized by the excretion of 7-substituted isomers of biopterin and neopterin and 7-oxo-biopterin in the urine of patients. It has been shown that the 7-substituted isomers of biopterin and neopterin derive from L-tetrahydrobiopterin and D-tetrahydroneopterin and are formed during hydroxylation of phenylalanine to tyrosine with rat liver dehydratase-free phenylalanine hydroxylase. We have now obtained identical results using human phenylalanine hydroxylase. The identity of the pterin formed in vitro and derived from L-tetrahydrobiopterin as 7-(1',2'-dihydroxypropyl)pterin was proven by gas-chromatography mass spectrometry. Tetrahydroneopterin and 6-hydroxymethyltetrahydropterin also are converted to their corresponding 7-substituted isomers and serve as cofactors in the phenylalanine hydroxylase reaction. Dihydroneopterin is converted by dihydrofolate reductase to the tetrahydro form which is biologically active as a cofactor for the aromatic amino acid monooxygenases. The 6-substituted pterin to 7-substituted pterin conversion occurs in the absence of pterin-4a-carbinolamine dehydratase and is shown to be a nonenzymatic process. 7-Tetrahydrobiopterin is both a substrate (cofactor) and a competitive inhibitor with 6-tetrahydrobiopterin (Ki approximately 8 microM) in the phenylalanine hydroxylase reaction. For the first time, the formation of 7-substituted pterins from their 6-substituted isomers has been demonstrated with tyrosine hydroxylase, another important mammalian enzyme which functions in the hydroxylation of phenylalanine and tyrosine.

7-Substituted pterins: formation during phenylalanine hydroxylation in the absence of dehydratase.

Previously we described a new form of human hyperphenylalaninemia characterized by the formation of 7-substituted pterins. We present evidence strongly suggesting that the 7-substituted pterins are formed by rearrangement of 6-substituted pterins. This rearrangement occurs during the phenylalanine hydroxylase reaction cycle which normally involves the enzymes phenylalanine hydroxylase, pterin-4a-OH-dehydratase, and q-dihydropterin reductase, specifically in the absence of dehydratase activity. We conclude that formation of 7-substituted pterins in humans is a consequence of an absence of dehydratase activity, which might result from a genetic defect. A chemical mechanism for this rearrangement is presented. Our results also suggest that tetrahydroneopterin can be a cofactor for the phenylalanine hydroxylase system in vivo.

7-Substituted pterins. A new class of mammalian pteridines.

Three novel pteridines have been isolated from the urine of patients with a new variant of 6-(L-erythro-1',2'-dihydroxypropyl)-5,6,7,8-tetrahydropterin (tetrahydrobiopterin) deficiency, showing hyperphenylalaninemia. From the results of high performance liquid chromatography, oxidative degradation, and gas chromatography-electron impact mass spectrometry, their structures were identified as 7-(D-erythro-1',2',3'-trihydroxypropyl)-pterin (7-neopterin), 7-(L-erythro-1',2'-dihydroxypropyl)-pterin (7-biopterin), and 6-oxo-7-(L-erythro-1',2'-dihydroxypropyl)-pterin (6-oxo-7-biopterin). The ratio of biopterin to 7-biopterin in the patients' urines was 1:1, and after oral loading with tetrahydrobiopterin, 7-biopterin excretion rose parallel to biopterin. This finding suggests that 7-substituted pterins may be formed endogenously by a yet unknown isomerization reaction. The cause of hyperphenylalaninemia is still unclear. The activities of the enzymes involved in tetrahydrobiopterin biosynthesis and regeneration were found to be normal in the patients, and no effect of 7-biopterin on these enzymes was observed in vitro. However, compared with the normal cofactor, tetrahydrobiopterin, the Km values of tetrahydro-7-biopterin for phenylalanine hydroxylase and dihydropteridine reductase are 20 and 5 times higher, respectively.

1H-NMR and mass spectrometric studies of tetrahydropterins. Evidence for the structure of 6-pyruvoyl tetrahydropterin, an intermediate in the biosynthesis of tetrahydrobiopterin.

The conversion of dihydroneopterin triphosphate in the presence of 6-pyruvoyl tetrahydropterin synthase was followed by 1H-NMR spectroscopy. The interpretation of the spectra of the product is unequivocal: they show formation of a tetrahydropterin system carrying a stereospecifically oriented substituent at the asymmetric C(6) atom. The spectra are compatible with formation of a (3')-CH3 function, and with complete removal of the 1' and 2' hydrogens of dihydroneopterin triphosphate. The fast-atom-bombardment/mass spectrometry study of the same product yields a [M + H]+ ion at m/z 238 compatible with the structure of 6-pyruvoyl tetrahydropterin. The data support the proposed structure of 6-pyruvoyl tetrahydropterin as a key intermediate in the biosynthesis of tetrahydrobiopterin.

Human liver 6-pyruvoyl tetrahydropterin reductase is biochemically and immunologically indistinguishable from aldose reductase.

6-Pyruvoyl tetrahydropterin reductase has been implicated in the biosynthesis of tetrahydrobiopterin. Using immunochemical and biochemical techniques the purified human liver enzyme was shown to be identical to aldose reductase. This suggests that 6-pyruvoyl tetrahydropterin reductase may play an additional role in the reduction of aldehydes derived from the biogenic amine neuro-transmitters and corticosteroid hormones as well as in the pathogenesis of diabetic complications, as has been postulated for aldose reductase.

Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from salmon liver.

Salmon liver was chosen for the isolation of 6-pyruvoyl tetrahydropterin synthase, one of the enzymes involved in tetrahydrobiopterin biosynthesis. A 9500-fold purification was obtained and the purified enzyme showed two single bands of 16 and 17 kDa on SDS/PAGE. The native enzyme (68 kDa) consists of four subunits and needs free thiol groups for enzymatic activity as was shown by reacting the enzyme with the fluorescent thiol reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The enzyme is heat-stable up to 80 degrees C, has an isoelectric point of 6.0-6.3, and a pH optimum at 7.5. The enzyme is Mg2+ -dependent and has a Michaelis constant for its substrate dihydroneopterin triphosphate of 2.2 microM. The turnover number of the purified salmon liver enzyme is about 50 times as high as that of the enzyme purified from human liver. It does not bind to the lectin concanavalin A, indicating that it is free of mannose and glucose residues. Polyclonal antibodies raised against the purified enzyme in Balb/c mice were able to immunoprecipitate enzyme activity. The same polyclonal serum was not able to immunoprecipitate enzyme activity of human liver 6-pyruvoyl tetrahydropterin synthase, nor was any cross-reaction in ELISA tests seen.