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Specificity of flavin adenine dinucleotide pyrophosphorylase for flavin phosphates and nucleoside triphosphates
Vol. 14, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Donald B. McCormick Graduate School of Nutrition Cornell University Ithaca, NewYork
Received
December
18,
1963
The biosynthesis
of flavin
adenine dinucleotide
tissues has been observed by several investigators
(FAD) in plant and animal (1).
The overall
mechanism
of FAD biosynthesis was elucidated by Schrecker and Kornberg (2) who partially purified
an enzyme from yeast which catalyzes
the reversible
and inorganic pyrophosphate from riboflavin-5'-phosphate 5'-triphosphate
(ATP).
synthesis of FAD
(EMN) and adenosine-
That a similar meansfor FAD synthesis occurs in
animal tissues was demonstrated by Watarai --et al (3) who found that liver preparations catalyze the incorporation of FMN (P 32) into FAD without loss of specific tic
radioactivity activity
of the phosphate.
for FAD in liver
plasm and is largely
DeLuca and Kaplan (4) showedthe synthe-
is located in the soluble fraction
maskedby FAD-destroying enzymes which can be partially
removed during purification
of the FAD pyrophosphorylase.
Heretofore a study has not been made on the specificity phorylase for both interacting triphosphate.
substrates, flavin
Since several riboflavin
analogues of FMN (5-‘7), of FAD should be sought. nucleoside triphosphates
phosphate and nucleoside
by flavokinase,
the conversion of the latter
able to support
are converted to
to functional
analogues
Moreover the presence of FAD pyrophosphorylase and in the soluble fraction
of the cell,
but lack of bio-
analogues of FAD in which the adenine moiety is replaced by other
purines or pyrimidines,
*
of FAD pyrophos-
analogues are partially
growth in organisms (1) and, via catalysis
synthetic
of the cyto-
would suggest essentially
Supported by Research Grant AM-04585
absolute specificity
for ATP
frcm the U.S. Public Health Service. 493
Vol. 14, No. 6, 1964
BIOCHEMICAL
and should. bear investigation. phosphorylase from rat liver
AND BIOPHYSICAL
The present study demonstrates that FAD pyroexhibits
a relative
phosphate and an apparent absolute specificity
specificity for ATP.
FMN, FAD, and ribo-and aeoxyribofuranoside-5'-triphosphates
of purines an& pyrimidines were the highest purity Isoriboflavin
C-. flavin
available
DohmeLaboratories. and D-Erythroflavin
64dethylriboflavin
alloxazine
were frcm the Merck Sharp ant
(6,7-dimethyl-9-(1'~D-erythrityl)isoalloxazine)
(8).
g-nitroanilines
were prepared
and aldoses by the method of
(6,7-dimethyl-g-(5'-hydroxypentyl)-
2’,3’,4’-Triaeoxyribaflavin
aa
isoalloxazine)
and Parabo-
(6-methyl-9-(1'~D-ribityl)isoalloxaeine)
corresponding substituted
Kuhn -et -* al
from Sigma Chemical
(5,6-dimethyl-9-(1'~D-ribityl)isoalloxazine
(6,7-dimethyl-g-(1'-D-arabityl)isoalloxazine)
from their
for the flavin
and Methods
Materials
Riboflavin,
RESEARCH COMMUNICATIONS
Z!'-deoxyglyceroflatin
(6,7-dimethyl-g-(3'~hydromropyl)iso-
were synthesized via displacement reactions with the appropriate
alcohol and 1,2-&methyl-4,5-dinitrobenzene
as described by Weygana et al.
The monophosphate ester of each of the flatins the terminal bydroxymethyl and Farkas (10).
amino. (9).
was prepared by phosphorylation
group with dichlorophosphoric
of
acid according to Flexsel
The FAD pyrophosphorylase from rat liver
was partially
purified
as described by DeLuca and Kaplan (4). Mixtures flavin
for assessing substrate reactivity
contained 0.5 I&¶ BMNor other
phosphate,1 d4 ATP or other nucleoside triphosphate,
potassium phosphate buffer
(pH 7.5),
and 2.5 mg. of protein
Incubation was carried out in the dark at 37' for 60 min. minated by heating the mixtures in a boiling
extracted
in 3 ml. total
with 0.25 ml. Of liquid
volume.
The reaction was ter-
water bath for 3 min.
were saturated with (14R4)2S04,the mixtures filtered, the filtrates
1 d4 MgC12, 25 mM
The solutions
and the flavin
(warmed) phenol.
compoundsin
50 Lsmbdaaliquo
of the phenol layer were analyzed for FAD or its analogues by the paper chromatographic method of Giri a solvent
system.
the paper strips
(ll),
with @utyl
alcohol:
acetic acid: water (4:1:5)
The chromatograms were examined under an ultraviolet containing FAD compoundswere cut out and eluted 494
light,
as
and
with 3 IIM potass
Vol. 14, No. 6, 1964
phosphate buffer, metrically
BIOCHEMICAL
pH 7.
AND BIOPHYSICAL
FAD ccqounds in the eluates were determined fluoro-
according to the method of Yagi (12).
and appropriate
RESEARCH COMMUNICATIONS
Flav-ins, flavin
phosphates,
standards also were applied to WhatmanNo. 1 paper and the fluor-
escent spots observed under ultraviolet
light
after
developnent of the chromato-
grams in ascending solvents as described by Huennekensand Felton (13). confirmation
of purity
and mobility
of the flavin
Secondary
compoundswas obtained in this
manner (14). Results and Discussion The
5?
values of flatins,
flavin
phosphates, and flavin
adenlne dinucleotides
are shown in Table I.
TARLEI Paper Chrcmatography of Flavin Compounds Compound
RF values in ascending solvents S % s2 0.30 0.30 0.80 0.53 0.12 0.17
Riboflavin t1 -5'-phosphate II adenine dinucleotide Isoriboflavin I, -5'-phosphate 3, adenine dinucleotide 644ethylriboflavin I, -5'~phosphate II adenine dinucleotide
0.35 0.28 0.50
6,7-Mbranoriboflavin~ I, +*-phosphate 11 adenine dinucleotide D-Araboflavin 9, -5'-phosphate II adenine dinucleotide D-Erythroflavin I, -k'-phosphate II adenine dinucleotide 2',3',4'-Trideoxyriboflavin ‘I +*-phosphate 11 adenine dinucleotide 2'Geoxyglyceroflavin 8, -3'~phosphate II adenine dinucleotide
0.05 0.30
0.23
0.11
0.79 0.17
0.40
0.05 0.25
0.80
0.64
0.09
0.19
0.18 0.45
0.44 0.17 0.06
0.70
0.31 0.60
0.32
0.80 0.20
0.33 0.64 0.32
0.56 0.25 0.57
0.04
0.12
0.05 0.38 0.15 0.05 0.65 0.23
0.07
0.57 0.18 0.06
0.19
0.86 0.28 0.95 0.35 0.w
0.31
= 5%aqueous NafIG04; S2 = n-butyl alcohol: acetic acid: * Solvents system: "I; ase); S3 E pheno : n-butyl alcohol: water (16:3:10, lower water (4:1:5, upper p phase). * !Chis flavin and its nucleotides exhibit a diminished fluorescence efficiency.
495
*
BIOCHEMICAL
Vol. 14, No. 6, 1964
Each of the dinucleotides
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
reported is observed as the enzymatic product in the
FAD pyrophosphorylase reaction with ATP and the respective substrates.
flatin
phosphate as
Thus the pyrophosphorylase has somecapacity for catalyzing
densation of the adenylate moiety of ATR with variously
substituted
ing an orthophosphate ester grouping on the side chain at position zine ring.
the con-
flavins
bear-
9 of the alloxr
However, when FM3 was included with other nucleoside triphosphates Ribo-and deoxyribofuranoside-
replacing ATP, no FAD analogues could be detected. 5'-triphosphates
with inosine,
were all inactive.
guanine, cytosine,
uracil,
This apparent absolute specificity
and thymine as the base
of FAD pyrophosphorylase
for AW would explain why no analogues in which adenine is replaced by other purines or pyrimidines
are found biosynthetically
nucleoside triphosphates The quantitative
even though both enzyme and
occur together in the soluble fraction
assays of enzymatically
of the cytoplasm.
synthesized flavin
adenine dinucleo-
tides are shown in Table II.
TABLE II Enzyme-Catalyzed Formation of Flavin Adenine Mnucleotides Flatin
monophosphateadded (500 pM)
Flav-in adenine dinucleotide hl4
Riboflavin-5'-phosphate Isoriboflavin-5'-phosphate 6+4ethylriboflatin+'-phosphate 6,7-Dibrwnoriboflavin-5'-phosphate D-Araboflavin-5'-phosphate D-Erythroflavin-4'-phosphate 2',3',4'-Trideoxyriboflavin-5'-phosphate 2'-Deoxyriboflavin-3'-phosphate
Under the assay conditions
10.3 28.8 7.5 3.2
1.2 1.2
2.2 1.0
employed, isoriboflavin-5'-phosphate
substrate than the natural one, FMN. As demonstrated earlier 5'-phosphate is not formed in the flavokinase normal phosphorylation is an effective
of riboflavin.
suggest that isoriboflavin
is an even better
(5,6),
isoriboflavin.
reaction and does not inhibit
the
Also it has been shown that isoriboflatin
antagonist to riboflavin
to TPNRcytochrome c reductase -in vitro
formed
in -- viva and inhibits (15).
may competitively 496
Taken altogether
the binding of lMN these findings
displace FMN, both as substrate for
Vol. 14, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FAD pyrophosphorylase and as coenzyme. The observed conversions of the 5'-phosphates of 6-methylribo-,6,7-dibromoribo-, is supported by their logical
activities
and D-araboflavins
known flavokinase-catalyzed
(1).
gation of the biological
phosphorylations
SFmilarly the formation of a flavin
from D-erythroflavin-4'-phosphate
to their
FAD analogues
(5-7) and bio-
adenine dinucleotide
is supported by the recent syntheses and investi-
activities
of this flavin
and its phosphate ester (16).
with @-hydroxyalkyl
side chains has not yet been
The reactivity
of the flavins
investigated
in other systems.
REIXRFJCES 1. 2.
3. 4. 2. 0.
Beinert, H., in P. D, Boyer, H. Lardy, and K. Myrback (Editors), The -- Enzymes, Vol. II, Academic Press, Inc., NewYork, 1960, p. 339. Schrecker, A. W., and Kornberg, A., 2. Biol, Chem., 182, 795 (1950). Watarai, K., Uchida, T., Ishikawa, E., Itaya,Tand Katsunm, N., Vitamins
(KYoto),
g, 331 (1955).
% 6 (1958). DeLuca, C., and Kaplan, N. O., Biochem. Biophys. Acta, -McCormick, D. B., 2. Biol. Chem., 237, 91591). McCormick, D. B., andxler, R., Bi;;chem. Biophys. Acta, 6& 326 (1962). Mccomick, D. B., Arsenis, C., and-Hamnerich, P., J. B&l. Chem.> .?38, 30% (1963 -z 1jE (1937). Kuhn, R., Vetter, H., and Rzeppa, H., Ber. &.-c&mxs., chs. BtZT, 84T iol (1951). Weygand, F., Lowenfeld, R., and Mollerx, Flexser, L. A., and Farkas, W. G., U. S. Patent 2xO;lF; Chem; Abstr.,s 12379 (1961). Giri, K. V., and Krishnaswamy, P. R., 2. Indian Inst. S&.., 38, 232 (1956). Yagi, K., 2. Biochem. (Tokyo), & 161 (1s Huennekens, F. M., and Felton, S. P., in S. P. Colowick and N. 0. Kaplan (Editors Methods in m Vol. III, Academic Press, Inc., NewYork, 1957, p. 957. Kirmnich,?. A., and McCormick, D. B., J. Chromate;+., in press (1963). Kearney, E. B., 2. Biol. Chem., 194, 767X1952). Uehara, K., Sugeno,r andizoguchi, T., J,. Biochem. (Tokyo), 5& 267 (1963).
87: 9.
10. 11. 12.
13. 14.
15. 16.
497
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Donald B. McCormick Graduate School of Nutrition Cornell University Ithaca, NewYork
Received
December
18,
1963
The biosynthesis
of flavin
adenine dinucleotide
tissues has been observed by several investigators
(FAD) in plant and animal (1).
The overall
mechanism
of FAD biosynthesis was elucidated by Schrecker and Kornberg (2) who partially purified
an enzyme from yeast which catalyzes
the reversible
and inorganic pyrophosphate from riboflavin-5'-phosphate 5'-triphosphate
(ATP).
synthesis of FAD
(EMN) and adenosine-
That a similar meansfor FAD synthesis occurs in
animal tissues was demonstrated by Watarai --et al (3) who found that liver preparations catalyze the incorporation of FMN (P 32) into FAD without loss of specific tic
radioactivity activity
of the phosphate.
for FAD in liver
plasm and is largely
DeLuca and Kaplan (4) showedthe synthe-
is located in the soluble fraction
maskedby FAD-destroying enzymes which can be partially
removed during purification
of the FAD pyrophosphorylase.
Heretofore a study has not been made on the specificity phorylase for both interacting triphosphate.
substrates, flavin
Since several riboflavin
analogues of FMN (5-‘7), of FAD should be sought. nucleoside triphosphates
phosphate and nucleoside
by flavokinase,
the conversion of the latter
able to support
are converted to
to functional
analogues
Moreover the presence of FAD pyrophosphorylase and in the soluble fraction
of the cell,
but lack of bio-
analogues of FAD in which the adenine moiety is replaced by other
purines or pyrimidines,
*
of FAD pyrophos-
analogues are partially
growth in organisms (1) and, via catalysis
synthetic
of the cyto-
would suggest essentially
Supported by Research Grant AM-04585
absolute specificity
for ATP
frcm the U.S. Public Health Service. 493
Vol. 14, No. 6, 1964
BIOCHEMICAL
and should. bear investigation. phosphorylase from rat liver
AND BIOPHYSICAL
The present study demonstrates that FAD pyroexhibits
a relative
phosphate and an apparent absolute specificity
specificity for ATP.
FMN, FAD, and ribo-and aeoxyribofuranoside-5'-triphosphates
of purines an& pyrimidines were the highest purity Isoriboflavin
C-. flavin
available
DohmeLaboratories. and D-Erythroflavin
64dethylriboflavin
alloxazine
were frcm the Merck Sharp ant
(6,7-dimethyl-9-(1'~D-erythrityl)isoalloxazine)
(8).
g-nitroanilines
were prepared
and aldoses by the method of
(6,7-dimethyl-g-(5'-hydroxypentyl)-
2’,3’,4’-Triaeoxyribaflavin
aa
isoalloxazine)
and Parabo-
(6-methyl-9-(1'~D-ribityl)isoalloxaeine)
corresponding substituted
Kuhn -et -* al
from Sigma Chemical
(5,6-dimethyl-9-(1'~D-ribityl)isoalloxazine
(6,7-dimethyl-g-(1'-D-arabityl)isoalloxazine)
from their
for the flavin
and Methods
Materials
Riboflavin,
RESEARCH COMMUNICATIONS
Z!'-deoxyglyceroflatin
(6,7-dimethyl-g-(3'~hydromropyl)iso-
were synthesized via displacement reactions with the appropriate
alcohol and 1,2-&methyl-4,5-dinitrobenzene
as described by Weygana et al.
The monophosphate ester of each of the flatins the terminal bydroxymethyl and Farkas (10).
amino. (9).
was prepared by phosphorylation
group with dichlorophosphoric
of
acid according to Flexsel
The FAD pyrophosphorylase from rat liver
was partially
purified
as described by DeLuca and Kaplan (4). Mixtures flavin
for assessing substrate reactivity
contained 0.5 I&¶ BMNor other
phosphate,1 d4 ATP or other nucleoside triphosphate,
potassium phosphate buffer
(pH 7.5),
and 2.5 mg. of protein
Incubation was carried out in the dark at 37' for 60 min. minated by heating the mixtures in a boiling
extracted
in 3 ml. total
with 0.25 ml. Of liquid
volume.
The reaction was ter-
water bath for 3 min.
were saturated with (14R4)2S04,the mixtures filtered, the filtrates
1 d4 MgC12, 25 mM
The solutions
and the flavin
(warmed) phenol.
compoundsin
50 Lsmbdaaliquo
of the phenol layer were analyzed for FAD or its analogues by the paper chromatographic method of Giri a solvent
system.
the paper strips
(ll),
with @utyl
alcohol:
acetic acid: water (4:1:5)
The chromatograms were examined under an ultraviolet containing FAD compoundswere cut out and eluted 494
light,
as
and
with 3 IIM potass
Vol. 14, No. 6, 1964
phosphate buffer, metrically
BIOCHEMICAL
pH 7.
AND BIOPHYSICAL
FAD ccqounds in the eluates were determined fluoro-
according to the method of Yagi (12).
and appropriate
RESEARCH COMMUNICATIONS
Flav-ins, flavin
phosphates,
standards also were applied to WhatmanNo. 1 paper and the fluor-
escent spots observed under ultraviolet
light
after
developnent of the chromato-
grams in ascending solvents as described by Huennekensand Felton (13). confirmation
of purity
and mobility
of the flavin
Secondary
compoundswas obtained in this
manner (14). Results and Discussion The
5?
values of flatins,
flavin
phosphates, and flavin
adenlne dinucleotides
are shown in Table I.
TARLEI Paper Chrcmatography of Flavin Compounds Compound
RF values in ascending solvents S % s2 0.30 0.30 0.80 0.53 0.12 0.17
Riboflavin t1 -5'-phosphate II adenine dinucleotide Isoriboflavin I, -5'-phosphate 3, adenine dinucleotide 644ethylriboflavin I, -5'~phosphate II adenine dinucleotide
0.35 0.28 0.50
6,7-Mbranoriboflavin~ I, +*-phosphate 11 adenine dinucleotide D-Araboflavin 9, -5'-phosphate II adenine dinucleotide D-Erythroflavin I, -k'-phosphate II adenine dinucleotide 2',3',4'-Trideoxyriboflavin ‘I +*-phosphate 11 adenine dinucleotide 2'Geoxyglyceroflavin 8, -3'~phosphate II adenine dinucleotide
0.05 0.30
0.23
0.11
0.79 0.17
0.40
0.05 0.25
0.80
0.64
0.09
0.19
0.18 0.45
0.44 0.17 0.06
0.70
0.31 0.60
0.32
0.80 0.20
0.33 0.64 0.32
0.56 0.25 0.57
0.04
0.12
0.05 0.38 0.15 0.05 0.65 0.23
0.07
0.57 0.18 0.06
0.19
0.86 0.28 0.95 0.35 0.w
0.31
= 5%aqueous NafIG04; S2 = n-butyl alcohol: acetic acid: * Solvents system: "I; ase); S3 E pheno : n-butyl alcohol: water (16:3:10, lower water (4:1:5, upper p phase). * !Chis flavin and its nucleotides exhibit a diminished fluorescence efficiency.
495
*
BIOCHEMICAL
Vol. 14, No. 6, 1964
Each of the dinucleotides
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
reported is observed as the enzymatic product in the
FAD pyrophosphorylase reaction with ATP and the respective substrates.
flatin
phosphate as
Thus the pyrophosphorylase has somecapacity for catalyzing
densation of the adenylate moiety of ATR with variously
substituted
ing an orthophosphate ester grouping on the side chain at position zine ring.
the con-
flavins
bear-
9 of the alloxr
However, when FM3 was included with other nucleoside triphosphates Ribo-and deoxyribofuranoside-
replacing ATP, no FAD analogues could be detected. 5'-triphosphates
with inosine,
were all inactive.
guanine, cytosine,
uracil,
This apparent absolute specificity
and thymine as the base
of FAD pyrophosphorylase
for AW would explain why no analogues in which adenine is replaced by other purines or pyrimidines
are found biosynthetically
nucleoside triphosphates The quantitative
even though both enzyme and
occur together in the soluble fraction
assays of enzymatically
of the cytoplasm.
synthesized flavin
adenine dinucleo-
tides are shown in Table II.
TABLE II Enzyme-Catalyzed Formation of Flavin Adenine Mnucleotides Flatin
monophosphateadded (500 pM)
Flav-in adenine dinucleotide hl4
Riboflavin-5'-phosphate Isoriboflavin-5'-phosphate 6+4ethylriboflatin+'-phosphate 6,7-Dibrwnoriboflavin-5'-phosphate D-Araboflavin-5'-phosphate D-Erythroflavin-4'-phosphate 2',3',4'-Trideoxyriboflavin-5'-phosphate 2'-Deoxyriboflavin-3'-phosphate
Under the assay conditions
10.3 28.8 7.5 3.2
1.2 1.2
2.2 1.0
employed, isoriboflavin-5'-phosphate
substrate than the natural one, FMN. As demonstrated earlier 5'-phosphate is not formed in the flavokinase normal phosphorylation is an effective
of riboflavin.
suggest that isoriboflavin
is an even better
(5,6),
isoriboflavin.
reaction and does not inhibit
the
Also it has been shown that isoriboflatin
antagonist to riboflavin
to TPNRcytochrome c reductase -in vitro
formed
in -- viva and inhibits (15).
may competitively 496
Taken altogether
the binding of lMN these findings
displace FMN, both as substrate for
Vol. 14, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FAD pyrophosphorylase and as coenzyme. The observed conversions of the 5'-phosphates of 6-methylribo-,6,7-dibromoribo-, is supported by their logical
activities
and D-araboflavins
known flavokinase-catalyzed
(1).
gation of the biological
phosphorylations
SFmilarly the formation of a flavin
from D-erythroflavin-4'-phosphate
to their
FAD analogues
(5-7) and bio-
adenine dinucleotide
is supported by the recent syntheses and investi-
activities
of this flavin
and its phosphate ester (16).
with @-hydroxyalkyl
side chains has not yet been
The reactivity
of the flavins
investigated
in other systems.
REIXRFJCES 1. 2.
3. 4. 2. 0.
Beinert, H., in P. D, Boyer, H. Lardy, and K. Myrback (Editors), The -- Enzymes, Vol. II, Academic Press, Inc., NewYork, 1960, p. 339. Schrecker, A. W., and Kornberg, A., 2. Biol, Chem., 182, 795 (1950). Watarai, K., Uchida, T., Ishikawa, E., Itaya,Tand Katsunm, N., Vitamins
(KYoto),
g, 331 (1955).
% 6 (1958). DeLuca, C., and Kaplan, N. O., Biochem. Biophys. Acta, -McCormick, D. B., 2. Biol. Chem., 237, 91591). McCormick, D. B., andxler, R., Bi;;chem. Biophys. Acta, 6& 326 (1962). Mccomick, D. B., Arsenis, C., and-Hamnerich, P., J. B&l. Chem.> .?38, 30% (1963 -z 1jE (1937). Kuhn, R., Vetter, H., and Rzeppa, H., Ber. &.-c&mxs., chs. BtZT, 84T iol (1951). Weygand, F., Lowenfeld, R., and Mollerx, Flexser, L. A., and Farkas, W. G., U. S. Patent 2xO;lF; Chem; Abstr.,s 12379 (1961). Giri, K. V., and Krishnaswamy, P. R., 2. Indian Inst. S&.., 38, 232 (1956). Yagi, K., 2. Biochem. (Tokyo), & 161 (1s Huennekens, F. M., and Felton, S. P., in S. P. Colowick and N. 0. Kaplan (Editors Methods in m Vol. III, Academic Press, Inc., NewYork, 1957, p. 957. Kirmnich,?. A., and McCormick, D. B., J. Chromate;+., in press (1963). Kearney, E. B., 2. Biol. Chem., 194, 767X1952). Uehara, K., Sugeno,r andizoguchi, T., J,. Biochem. (Tokyo), 5& 267 (1963).
87: 9.
10. 11. 12.
13. 14.
15. 16.
497