50I
S-17-3
7-Substituted
Pterins:
H.-CH.CURTIUS,
Formation
C.ADLER,
.REBRIN,
(*),
ana
and
Occurence
C.HEIZMANN,
N.BLAU,
.UHISLA
(**)I
Deptartment of Pediatrics, Division of Clinical Chemistriy, University of Zurich, Zurich, Switzerland; (*) Institute for Biochemical Human Genetics, University of Greifswald, Greifswald, FRG; (**) Faculty of Biology, University of Konstanz, D-7750 Konstanz FRG INTRODUCTION Three novel pterins, 7-iso-biopterin (primapterin), 7-iso-neopterin (anapterin) and 6-oxo-primapterin have been isolated from urine of patients with a new variant of BH4 deficiency, showing transient hyperphenylalaninemia 1,2 By means of GC-MS, oxydation to the 7-carboxylic acids, and comparison with reference substances the structure of the above mentioned compounds could be proven 3 In order to get more information about the ratios of 7-biopterin and 7neopterin relative to the 6-isomers, we analyzed normal human urine as well as the urines of classical and atypical PKU patients for the occurence of the 7substituted isomers by means of mass fragmentography and HPLC. It could be shown that both 7-iso compounds are present in every human urine, blood and in the liver of man and mouse 3,4. GC-MS-selected ion monitoring was performed under negative chemical ionisation conditions for the analysis of these unusual metabolites in urine and tissues. The concentration of primapterin in those patients is app. 1:1 compared to biopterin and that of anapterin about 4%. In normal controls the excretion of the unusual metabolites are only about 4% each for both primapterin and anapterin in comparison with the 6-isomers. The excretion of 7-substituted pterins appears to be associated with an enzyem defect in the aromatic amino acid hydroxylating system. Formation of 7-pterins was also accomplished in vitro when the phenylalanine hydroxylase reactin (using human or rat enzyme) was carried out with 4a-carbinolamine dehydratase free preparations. The 7-substituted pterins are formed from the 6-substituted ones 3,5,6,7. The conclusion was reached that the patients suffering from this special form of hyperphenylalaninemia most probably suffer from a carbinolamine dehydratase (PCDH) deficiency. During the hydroxylation of phenylalanine to tyrosine L-BH4 is converted to its 4a-OH-carbinolamine. A dehydration of this intermediate spezies to quinonoid dehydrobiopternin (BH2) is accomplished by 4a-carbinolamine dehydratase (PCDH) 8. In the absence of the PCDA activity 4a-carbinolamine accumulates. The carbinolamine is relativly stable at alkaline pH and in the absence of PCDH. These conditions favor accumulation of 4a-OH-carbinolamine in vitro and rearrangement to 7-pterins. The latter most probably occurs via a spiro intermediate 6. When D-NH4 was used instead of L-BH4 as reduced pterin cofactor in the in vitro experiments carried out with phenylalanine hydroxylase we observe the formation of the isomer 7-neopterin. Since 7-neopterin excretion was found also in healthy controls as well as in patients with classical and atypical PKUwe can conclude that tetrahydroneopterin is a cofactor also in vivo of the
•
Symposium (17)
502
Biopterin
aromatic amino acid hydroxylating system. In order to be used in this reactio dihydroneopterin first has to be formed from dihydroneopterin triphosphate an then must be reduced t tetrahydroneopterin by dihydrofolate reductase. In this paper w provide direct GC-MS evidence for formed 7-biopterin and its assumed mechanism of explanation for the hyperphenylalaninemia of these further aim was to find a reason for the occurence of patients and controls by verifying the hypothesis is asubstratefordihydrofolate reductase, and its of the pterin dependent hydroxylases in vivo. Also we have investigated whether the formation of 6-hydroxytetrahydromethylpterin is used instead of
the structure of in vitro formation. We also give a primapterinuric patients. A of 7-neopterin in the urine that D-dihydroneopterin tetrahydro form a cofactor 7-isomers BH4.
also
occurs
when
Methods: The pterins, previously oxidized with manganese dioxide under acidic conditions, were measured by HPLC with fluorescence detection (excitation/emission wavelengths were 350/450 nm).Inhibition experiments with dehydratase-free rat liver phenylalanine hydroxylase were performed in 0.1 M Tris-HCI, with 0.35 U enzyme (specific activity 8.5 U/g protein), 1 mM Lphenylalanine, and an excess of both catalase and DHPR, 5mM NADH, Ltetrahydrobiopterin (L-BH4) as substrate (concentrations from 5 to 20 uM) and L-7-BH4 as potential inhibitor (concentrations from 2 to 30 uM). For GC-MS measurements of the in vitro formed primapterin we used the method of Kuster et al. 9.
Figure.
The
metabolic
pathway
of
phenylalanine
hydroxylase
reaction
H.-CH.CURTIUS
et
al.
503
FORMATION OF 7-SUBST.PTERINS The biochemical origin of 7-biopterin and 7-neopterin in patients with transient hyperphenylalaninemia could not be explained over a long period of time. Only recently the biochemical pathway for their formation has been proposed 6,10,7. It is of special interest that after loading those patients with BH4 not only an increase of urinary exrection of biopterin but also of primapterin was observed 3. This observation and Viscontini's finding that the stereochemistry of the side chain of biopterin and primapterin is the same 5, is in agreement with the proposal that 7-pterins originate from their 6isomers.
INHIBITION STUDIES To explain the observed transient hyperphenylalaninemia in those patients one might assume that the 7-pterins inhibit the phenylalanine hydroxylase activity and this may be the reason for the increase of plasma phenylalanine. We have investigated this aspect and the results clearly indicate that 7-BH4 competes with 6-BH4 for the active site of the enzyme. The Ki value of 8 uM obtained for 7-L-BH4 compares to Km values of 20 uM and 1.5 uM, respectively, for 7-BH4 and 6-BH4 as substrates. This observation strongly suggests that in these patients phenylalanine hydroxylase inhibition by their own abnormal metablites, the 7-tetrahydrobiopterins, causes or contributes to hyperphenylalaninemia. In contrast no apparent inhibition of DHPR was found.
TETRAHYDRONEOPTERIN AS COFACTOR IN VIVO Although it is difficult to explain the occurence of hyperphenylalaninemia in these PKU patients in the early stage of can hypothesize that 7-BH4 and other pterins temporarily play cofactors and that the need for such cofactors varies at different development. We found substancial concentrations of tetrahydroneopterin incubation of D-dihydroneopterin with dihydrofolate reductase. Since tetrahydroneopterin is a cofactor for phenylalanine hydroxylase, and of the fact, that the activity is only 20% compared with BH4, this might play an important role in patients with 6-pyruvoyl-tetrahydropterin synthase deficiency. As these patients excrete increased amounts of but very low levels of biopterin D-NH4 might thus act as cofactor aromatic amino acid hydroxylases. This might be a possible explanation observation that these patients have low but nevertheless substantial of neurotransmitters in tissues and bodyfluids.
transient life, one roles as stages of after in vitro in spite compound neopterin for the for the levels
GC-MSEVIDENCEFOR PRIMAPTERIN To confirm the identity of primapterin produced during the in vitro incubation of 6-BH4 with phenylalanine hydroxylase free of 4a-carbinolamine dehydratase (PCDH) we used GC-MS. The silk] derivatives of the two isomers are clearly separated by GC. The mass spectra obtained from the two peaks were essentially identical, as expected for such isomers. These results were confirmed using authentic 7-and 6-biopterins. The fragmentation pattern obtained from primapterin confirms the presence of a pterin derivative, deduced from m/z 409, which corresponds to the pteridine nucleus fragment when the 11-2' bon is cleaved and a proton of the 3'-methyl group shifts to the 7-position of th pyrazine ring 9.The GC-MS data obtained of in vitro formed primapterin give
Symposium (17)
504
conclusive
evidence
for
the
proposed
Biopterin
structure.
INCUBATION STUDIES WITH 6-HYDROXYMETHYL TETRAHYDROPTERIN Using 6-hydroxymethyl tetrahydropterin instead of BH4 for the in vitro incubation experiments with phenylalanine hydroxylase free of PCDH we observed also formation of 7-hydroxymethyl tetrahydropterin. This observation suggests that the conversion of 6 to 7-isomers via the carbinolamine is a general process during the phenylalanine hydroxylase reaction.
CONCLUSION The patients with primapterinuria most probably suffer from PCDH. Conclusive information about the in vivo situation in primapterinuric patients, including those suffering from the suspected pterin-4a-carbinolamine dehydratase deficiency, can only be obtained by measuring PCDH activity directly in the tissue of the patients. Based on our recent results the pathway of the phenylalanine hydroxylase reaction and the role of the enzymes involved can be formulated as shown in the Figure.
ACKNOWLEDGEMENTS
This work was supported project No. 31-28797.90.
financially
by the
Swiss
National
Science
Foundation,
REFERENCES
1
2
3 4
5 6 7 8 9 10
Curtius,H.-Ch., Kuster,T., Matasovic,A., Blau,N. and Dhondt,J.-L. (1988) Primapterin, anapterin, and 6-oxo-primapterin, three new 7-substituted pterins identified in a patient with hyperphenylalaninemia. Biochem. Biophys.Res.Commun. 153, 715-721. Dhondt,J.-L., Guibaud,P., Rolland,M.O., Dorche,C., Andre,S. Forzy,G. and Hayte,J.M. (1988) Neonatal hyperphenylalaninemia presumably caused by a new variant of biopterin synthetase deficiency. Eur.J.Pediatr. 147, 153157. Curtius,H.-Ch., Kuster,T., Matasovic,A., Schoedon,G., Dhondt,J.L., Guibaud,T. and Blau,N. (1990) 7-Substituted pterins. A new class of mammalian pteridines. J.Biol.Chem. 265, 3923-3930. Curtius,H.-Ch., Adler,C., Matasovic,A. and ;mono" (1989) 7-substitute pterins, a new class of mammalian pteridines. In: Curtius,H.-Ch., Ghisl S. and Blau,N. (eds) Chemistry and Biology of Pteridines. Walter de Gruyter, Berlin, pp.274-285. Viscontini,M. (1990) Natural primapterin belongs to the L-series of 7(polyhydroxypropyl)pterins. Helv.Chim.Acta 73, 1064-1067. Curtius,H.-Ch., Adler,C., Rebrin,I., Heizmann,C.W. and Ghisla,S. (1990) 7-Substituted pterins: formation during phenylalanine hydroxylation in the absence of dehydratase. Biochem.Biophys.Res.Commun. 172, 1060-1066. Davis,M.D., Kaufmann,S. and Milstien,S. (1991) Conversion of 6-subsituted tetrahydropterins to 7-isomers via phenylalanine hydroxylase-generated intermediates. Proc.Natl.Acad.Sci.USA 88, 385-389. Lazarus,R.A., Benkovic,J.S. and Kaufman,S. (1983)J.Biol.Chem. 258, 10960-10962. Kuster,T., Niederwieser,A. (1983) Gas chromatrography-mass spectrometry of trimethylsilyl pteridines. J. Chromatogr. 278, 245-254. Davis,M.D. and Kaufman,S. (1991) 7-Tetrahydrobiopterin is an uncoupled cofactor for rat hepatic phenylalanine hydroxylase FEBS Letters 285, 17-20
S-17-3
7-Substituted
Pterins:
H.-CH.CURTIUS,
Formation
C.ADLER,
.REBRIN,
(*),
ana
and
Occurence
C.HEIZMANN,
N.BLAU,
.UHISLA
(**)I
Deptartment of Pediatrics, Division of Clinical Chemistriy, University of Zurich, Zurich, Switzerland; (*) Institute for Biochemical Human Genetics, University of Greifswald, Greifswald, FRG; (**) Faculty of Biology, University of Konstanz, D-7750 Konstanz FRG INTRODUCTION Three novel pterins, 7-iso-biopterin (primapterin), 7-iso-neopterin (anapterin) and 6-oxo-primapterin have been isolated from urine of patients with a new variant of BH4 deficiency, showing transient hyperphenylalaninemia 1,2 By means of GC-MS, oxydation to the 7-carboxylic acids, and comparison with reference substances the structure of the above mentioned compounds could be proven 3 In order to get more information about the ratios of 7-biopterin and 7neopterin relative to the 6-isomers, we analyzed normal human urine as well as the urines of classical and atypical PKU patients for the occurence of the 7substituted isomers by means of mass fragmentography and HPLC. It could be shown that both 7-iso compounds are present in every human urine, blood and in the liver of man and mouse 3,4. GC-MS-selected ion monitoring was performed under negative chemical ionisation conditions for the analysis of these unusual metabolites in urine and tissues. The concentration of primapterin in those patients is app. 1:1 compared to biopterin and that of anapterin about 4%. In normal controls the excretion of the unusual metabolites are only about 4% each for both primapterin and anapterin in comparison with the 6-isomers. The excretion of 7-substituted pterins appears to be associated with an enzyem defect in the aromatic amino acid hydroxylating system. Formation of 7-pterins was also accomplished in vitro when the phenylalanine hydroxylase reactin (using human or rat enzyme) was carried out with 4a-carbinolamine dehydratase free preparations. The 7-substituted pterins are formed from the 6-substituted ones 3,5,6,7. The conclusion was reached that the patients suffering from this special form of hyperphenylalaninemia most probably suffer from a carbinolamine dehydratase (PCDH) deficiency. During the hydroxylation of phenylalanine to tyrosine L-BH4 is converted to its 4a-OH-carbinolamine. A dehydration of this intermediate spezies to quinonoid dehydrobiopternin (BH2) is accomplished by 4a-carbinolamine dehydratase (PCDH) 8. In the absence of the PCDA activity 4a-carbinolamine accumulates. The carbinolamine is relativly stable at alkaline pH and in the absence of PCDH. These conditions favor accumulation of 4a-OH-carbinolamine in vitro and rearrangement to 7-pterins. The latter most probably occurs via a spiro intermediate 6. When D-NH4 was used instead of L-BH4 as reduced pterin cofactor in the in vitro experiments carried out with phenylalanine hydroxylase we observe the formation of the isomer 7-neopterin. Since 7-neopterin excretion was found also in healthy controls as well as in patients with classical and atypical PKUwe can conclude that tetrahydroneopterin is a cofactor also in vivo of the
•
Symposium (17)
502
Biopterin
aromatic amino acid hydroxylating system. In order to be used in this reactio dihydroneopterin first has to be formed from dihydroneopterin triphosphate an then must be reduced t tetrahydroneopterin by dihydrofolate reductase. In this paper w provide direct GC-MS evidence for formed 7-biopterin and its assumed mechanism of explanation for the hyperphenylalaninemia of these further aim was to find a reason for the occurence of patients and controls by verifying the hypothesis is asubstratefordihydrofolate reductase, and its of the pterin dependent hydroxylases in vivo. Also we have investigated whether the formation of 6-hydroxytetrahydromethylpterin is used instead of
the structure of in vitro formation. We also give a primapterinuric patients. A of 7-neopterin in the urine that D-dihydroneopterin tetrahydro form a cofactor 7-isomers BH4.
also
occurs
when
Methods: The pterins, previously oxidized with manganese dioxide under acidic conditions, were measured by HPLC with fluorescence detection (excitation/emission wavelengths were 350/450 nm).Inhibition experiments with dehydratase-free rat liver phenylalanine hydroxylase were performed in 0.1 M Tris-HCI, with 0.35 U enzyme (specific activity 8.5 U/g protein), 1 mM Lphenylalanine, and an excess of both catalase and DHPR, 5mM NADH, Ltetrahydrobiopterin (L-BH4) as substrate (concentrations from 5 to 20 uM) and L-7-BH4 as potential inhibitor (concentrations from 2 to 30 uM). For GC-MS measurements of the in vitro formed primapterin we used the method of Kuster et al. 9.
Figure.
The
metabolic
pathway
of
phenylalanine
hydroxylase
reaction
H.-CH.CURTIUS
et
al.
503
FORMATION OF 7-SUBST.PTERINS The biochemical origin of 7-biopterin and 7-neopterin in patients with transient hyperphenylalaninemia could not be explained over a long period of time. Only recently the biochemical pathway for their formation has been proposed 6,10,7. It is of special interest that after loading those patients with BH4 not only an increase of urinary exrection of biopterin but also of primapterin was observed 3. This observation and Viscontini's finding that the stereochemistry of the side chain of biopterin and primapterin is the same 5, is in agreement with the proposal that 7-pterins originate from their 6isomers.
INHIBITION STUDIES To explain the observed transient hyperphenylalaninemia in those patients one might assume that the 7-pterins inhibit the phenylalanine hydroxylase activity and this may be the reason for the increase of plasma phenylalanine. We have investigated this aspect and the results clearly indicate that 7-BH4 competes with 6-BH4 for the active site of the enzyme. The Ki value of 8 uM obtained for 7-L-BH4 compares to Km values of 20 uM and 1.5 uM, respectively, for 7-BH4 and 6-BH4 as substrates. This observation strongly suggests that in these patients phenylalanine hydroxylase inhibition by their own abnormal metablites, the 7-tetrahydrobiopterins, causes or contributes to hyperphenylalaninemia. In contrast no apparent inhibition of DHPR was found.
TETRAHYDRONEOPTERIN AS COFACTOR IN VIVO Although it is difficult to explain the occurence of hyperphenylalaninemia in these PKU patients in the early stage of can hypothesize that 7-BH4 and other pterins temporarily play cofactors and that the need for such cofactors varies at different development. We found substancial concentrations of tetrahydroneopterin incubation of D-dihydroneopterin with dihydrofolate reductase. Since tetrahydroneopterin is a cofactor for phenylalanine hydroxylase, and of the fact, that the activity is only 20% compared with BH4, this might play an important role in patients with 6-pyruvoyl-tetrahydropterin synthase deficiency. As these patients excrete increased amounts of but very low levels of biopterin D-NH4 might thus act as cofactor aromatic amino acid hydroxylases. This might be a possible explanation observation that these patients have low but nevertheless substantial of neurotransmitters in tissues and bodyfluids.
transient life, one roles as stages of after in vitro in spite compound neopterin for the for the levels
GC-MSEVIDENCEFOR PRIMAPTERIN To confirm the identity of primapterin produced during the in vitro incubation of 6-BH4 with phenylalanine hydroxylase free of 4a-carbinolamine dehydratase (PCDH) we used GC-MS. The silk] derivatives of the two isomers are clearly separated by GC. The mass spectra obtained from the two peaks were essentially identical, as expected for such isomers. These results were confirmed using authentic 7-and 6-biopterins. The fragmentation pattern obtained from primapterin confirms the presence of a pterin derivative, deduced from m/z 409, which corresponds to the pteridine nucleus fragment when the 11-2' bon is cleaved and a proton of the 3'-methyl group shifts to the 7-position of th pyrazine ring 9.The GC-MS data obtained of in vitro formed primapterin give
Symposium (17)
504
conclusive
evidence
for
the
proposed
Biopterin
structure.
INCUBATION STUDIES WITH 6-HYDROXYMETHYL TETRAHYDROPTERIN Using 6-hydroxymethyl tetrahydropterin instead of BH4 for the in vitro incubation experiments with phenylalanine hydroxylase free of PCDH we observed also formation of 7-hydroxymethyl tetrahydropterin. This observation suggests that the conversion of 6 to 7-isomers via the carbinolamine is a general process during the phenylalanine hydroxylase reaction.
CONCLUSION The patients with primapterinuria most probably suffer from PCDH. Conclusive information about the in vivo situation in primapterinuric patients, including those suffering from the suspected pterin-4a-carbinolamine dehydratase deficiency, can only be obtained by measuring PCDH activity directly in the tissue of the patients. Based on our recent results the pathway of the phenylalanine hydroxylase reaction and the role of the enzymes involved can be formulated as shown in the Figure.
ACKNOWLEDGEMENTS
This work was supported project No. 31-28797.90.
financially
by the
Swiss
National
Science
Foundation,
REFERENCES
1
2
3 4
5 6 7 8 9 10
Curtius,H.-Ch., Kuster,T., Matasovic,A., Blau,N. and Dhondt,J.-L. (1988) Primapterin, anapterin, and 6-oxo-primapterin, three new 7-substituted pterins identified in a patient with hyperphenylalaninemia. Biochem. Biophys.Res.Commun. 153, 715-721. Dhondt,J.-L., Guibaud,P., Rolland,M.O., Dorche,C., Andre,S. Forzy,G. and Hayte,J.M. (1988) Neonatal hyperphenylalaninemia presumably caused by a new variant of biopterin synthetase deficiency. Eur.J.Pediatr. 147, 153157. Curtius,H.-Ch., Kuster,T., Matasovic,A., Schoedon,G., Dhondt,J.L., Guibaud,T. and Blau,N. (1990) 7-Substituted pterins. A new class of mammalian pteridines. J.Biol.Chem. 265, 3923-3930. Curtius,H.-Ch., Adler,C., Matasovic,A. and ;mono" (1989) 7-substitute pterins, a new class of mammalian pteridines. In: Curtius,H.-Ch., Ghisl S. and Blau,N. (eds) Chemistry and Biology of Pteridines. Walter de Gruyter, Berlin, pp.274-285. Viscontini,M. (1990) Natural primapterin belongs to the L-series of 7(polyhydroxypropyl)pterins. Helv.Chim.Acta 73, 1064-1067. Curtius,H.-Ch., Adler,C., Rebrin,I., Heizmann,C.W. and Ghisla,S. (1990) 7-Substituted pterins: formation during phenylalanine hydroxylation in the absence of dehydratase. Biochem.Biophys.Res.Commun. 172, 1060-1066. Davis,M.D., Kaufmann,S. and Milstien,S. (1991) Conversion of 6-subsituted tetrahydropterins to 7-isomers via phenylalanine hydroxylase-generated intermediates. Proc.Natl.Acad.Sci.USA 88, 385-389. Lazarus,R.A., Benkovic,J.S. and Kaufman,S. (1983)J.Biol.Chem. 258, 10960-10962. Kuster,T., Niederwieser,A. (1983) Gas chromatrography-mass spectrometry of trimethylsilyl pteridines. J. Chromatogr. 278, 245-254. Davis,M.D. and Kaufman,S. (1991) 7-Tetrahydrobiopterin is an uncoupled cofactor for rat hepatic phenylalanine hydroxylase FEBS Letters 285, 17-20
