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α-Ketoglutarate and hydroxylation of γ-butyrobetaine
PRELIMINARY NOTES
503
BBA 2 1 2 2 0
~-Ketoglutarate and hydroxylation of ~-butyrobetaine Recently it has been reported that c~-ketoglutarate stimulates four different hydroxylation reactions, i.e. the hydroxylation of peptidyl proline to 4-hydroxyproline 1-3, the hydroxylation of 7-butyrobetaine to carnitine 4,5, the hydroxylation of thymine to 7-hydroxymethyluracil~, and the hydroxylation of peptidyl lysine to 5-hydroxylysineT,8. The same enzyme might hydroxylate both peptidyl proline and peptidyl lysine 7. The hydroxylases have common characteristics, since they all require molecular oxygen, ascorbate and Fe ~+. Pyruvate and oxaloacetate could replace ~-ketoglutarate in the hydroxylation of peptidyl proline, although they were much less effectiveLa. HUTTON,TAPPEL AND UDENFRIEND 3 found no relation between the hydroxylation of peptidyl proline and the disappearance of ~-ketoglutarate, which was ascribed mainly to the presence of transaminases in the crude enzyme preparation. It was suggested by these authors that ~-ketoglutarate might act as an allosteric effector, although a role as a cosubstrate could not be excluded from their experimental data. We have further studied the role of ~-ketoglutarate in the hydroxylation of 7-butyrobetaine by an enzyme from Pseudomonas sp. AK i (ref. 9). The enzyme has been obtained in soluble form by sonication of bacteria which had grown on 7-butyrobetaine as the sole source of carbon and nitrogen and it has been partially purified by chromatography on DEAE-cellulose and on hydroxylapatite. The partially purified enzyme has an absolute requirement for ascorbate, Fe 2+ and for ~-ketoglutarate 5. Several other organic acids have been tested fol their ability to replace ~-ketoglutarate but with negative results. Among others, fumarate, oxaloacetate, pyruvate, ~hydroxyglutarate, ~-keto-y-hydroxyglutarate and c~-ketoadipate have been tested. The change in ~-ketoglutarate concentration during hydroxylation of 7-butyrobetaine has been studied by enzymatic determinations of ~-ketoglutarate with L-glutamate: NAD + oxidoreductase (deaminating) (EC 1.4.1.2 ) obtained from C. F. Boehringer and Soehne GmbH, Mannheim, West Germany. In the assay for ~-ketoglutarate, the disappearance of NADH and also the simultaneous formation of NAD + have been determined by the spectrophotofluorometric methods of KAPLAN,COLOWICK AND BARNES10 and LOWRY, ROBERTS AND KAPPHAHN 11. A stoichiometric relationship has been observed between the formation of carnitine and the disappearance of ~-ketoglutarate in several experiments with varying concentrations of enzyme, ~-ketoglutarate and 7-butyrobetaine. In the presence of high concentrations of enzyme and 7-butyrobetaine, ~-ketoglutarate has been found to be limiting for the yield of hydroxylated product. In the absence of 7-butyrobetaine, there was no change in the concentration of c~-ketoglutarate during incubation with enzyme and other cofactors. Fig. I shows the iesults of an experiment with varying amounts of ~-ketoglutarate and correlates the formation of carnitine with the disappearance of ~-ketoglutarate. To verify the degradation of cc-ketoglutarate, incubations were carried out with E5-1~C~-ketoglutarate obtained from the Radiochemical Centre, Amersham, England. The reaction mixtures were first chromatographed on columns of silicic acid prepared as described by PRIOR I~'ERRAZAND RELVAS12. Fig. 2 shows a typical chromatogram Biochim. Biophys. Acta, 158 (1968) 5o3-5o5
504
PRELIMINARY NOTES
with two peaks containing radioactive material coinciding with ~-ketoglutaric acid and succinic acid, which had been added as carriers immediately before chromatography. The identity of the reaction product with succinic acid has been further established by chromatography on Dowex 2-X8 (2oo-4oo mesh; eluted with o.5 M 1.5
i
i
//
/Ib / / /@
1.C E 3.
@/ /
F
/
E
/ 0
/
/
/
/
1200
24
800
16
/
/
._c E
@// /
/
a 05 "6
2
/
E :3-
E
@// z"
400
I 1 0.5
I 1.0
1.5
Disappearance of 2-ketoglutarate, #moles
%
10
20 Fraction n u m b e r
3o~~ l
Fig. I. R e l a t i o n s h i p between the h y d r o x y l a t i o n of 7 - b u t y r o b e t a i n e to carnitine and the disa p p e a r a n c e of ~-ketoglutarate. The enzyme p r e p a r a t i o n had been prepared by sonication of P s e u d o m o n a s sp. A K I, centrifugation at 75ooo × g, s t r e p t o m y c i n precipitation, filtration t h r o u g h S e p h a d e x G-25, adsorption onto a c o l u m n of h y d r o x y l a p a t i t e and elution of the enzymatic activity with 0.05 M p o t a s s i u m p h o s p h a t e at p H 7.0. The incubation m i x t u r e contained: [rnethyl-t4C]7b u t y r o b e t a i n e , 2 ~moles; a-ketoglutarate, o. 4 #mole; sodium ascorbate, IO/,moles; FeSO 4. 7 H20, o. 4 p mole; catalase, I mg; nicotinamide, 4 ° / t m o l e s ; KC1, 2 5 / , m o l e s ; p o t a s s i u m p h o s p h a t e buffer at p H 7.o, 25/nnoles. The final volume was o. 7 ml. The incubations were carried o u t at 37 ° for 45 nlin. The formation of carnitine was determined by c h r o m a t o g r a p h y on columns of Dowex 5 ° as described in detail previously 14. The disappearance of a-ketoglutarate was determined by enzymatic assay at the end of the incubation period (see text). Fig. 2. C h r o m a t o g r a p h y on a column of silicic acid of the incubation m i x t u r e from enzymatic h y d r o x y l a t i o n of 7 - b u t y r o b e t a i n e to carnitine in the presence of F5-14C]~-ketoglutarate. F o r conditions of i n c u b a t i o n see legend to Fig. i. The c o l u m n was prepared from 8 g of silicic acid (Mallinckrodt, IOO mesh) and eluted with benzene-tert.-butanol (9:1, v/v) in 4-ml fractions. ~-Ketoglutaric acid (IO ing) and succinic acid (I rag) had been added before c h r o m a t o g r a p h y . The isotope c o n t e n t was determined b y counting of dried aliquots in a windowless gas-flow counter, and the r e m a i n d e r of the fractions was titrated with o.o2 M NaOH.
formic acid) and on paper (system 5 of ref. 12). Furthermore the product has been recrystallized to constant specific radioactivity with authentic succinic acid. Thus, the experiments have demonstrated an intimate coupling between the hydroxylation of 7-butyrobetaine to carnitine and the degradation of ~-ketoglutarate. Fumarate activates dopamine/3-hydroxylase, 3,4-dihydloxyphenylethylamine, ascorbate:oxygen oxidoreductase (hydroxylating) (EC I.I4.2.I), which is a copper enzyme requiring ascorbate. FRIEDMAN"AND KAUFMAN13 have suggested that in this case fumarate acts either by combining with the copper ion in a way which facilitates the oxidation of Cu + to Cu 2+ or by changing the conformation of the protein, thereby enchancing the reactivity of Cu +. Apparently, fumarate is not stoichiometrically converted to any other compound during the hydroxylation reaction 1~. Fumarate is not obligatory in the case of dopamine/3-hydroxylase in contrast to ~-ketoglutarate, which is required by peptidyl-proline hydroxylase, 7-butyrobetaine hydroxylase and thymine 7-hydroxylase. It appears unlikely from present data that the effect of Biochim. Biophys. Aria, 158 (1968) 503-505
PRELIMINARY NOTES
505
a-ketoglutarate in these reactions is that of an allosteric effector. One can only speculate upon the possibilities that a-ketoglutarate is responsible for the generation of reducing equivalents or that it is directly involved in the hydroxylation reaction, the degradation occurring as a consequence of the electron shifts during the hydroxylase reaction. This work has been supported by a grant from the Swedish Medical Research Council (I3X-585).
Department of Physiological Chemistry, Chemical Centre, University of Lund, Lund (Sweden)
GORAN LINDSTEDT SVEN LINDSTEDT BIRGIT 0 L A N D E R MARIANNE TOFFT
I J. J. HUTTON, JR., A. L. TAPPEL AND S. UDENFRIEND, Biochem. Biophys. Res. Commun., 2 4 (1966) 179. 2 K. I. KIVlRIKKO AND D. J. PROCKOP, Arch. Biochem. Biophys., 118 (1967) 611. 3 J. J. HUTTON, JR., A. L. TAPPEL AND S. UDENFRIEND, Arch. Biochem. Biophys., 118 (1967) 231. 4 G. LINOSTEDT, Dissertation, Karolinska I n s t i t u t e t , Stockholm, 1967. 5 G. LINDSTEDT, S. LINDSTEDT, T. MIDTVEDT AND M. TOFFT, Bioehem. J., lO 3 (1967) i9P. 6 M. T. ABBOTT, E. K. SCHANDL, R. F. LEE, T. S. PARKER AND R. J. MIDGETT, Biochim. Biophys. Acta, 132 (1967) 525 . 7 K. I. KIVIRIKKO AND D. J. PROCKOP, Proc. Natl. Acad. Sci. U.S., 57 (1967) 782. 8 E. HAUSMANN, Biochim. Biophys. Acta, 133 (1967) 591. 9 G. LINDSTEDT, S. LINDSTEDT, T. MIDTVEDT AND M. TOFFT, Biochemistry, 6 (1967) 1262. IO N. O. KAPLAN, S. P. COLOWICK AND C. C. BARNES, J. Biol. Chem., 191 (1951) 461. I I O. H. LOWRY, N. R. ROBERTS AND J. I. KAPPHAHN, J. Biol. Chem., 224 (1957) lO47. 12 F. G. PRIOR FERRAZ AND M. E. RELVAS, Clin. Chim. Acta, i i (1965) 244. 13 S. FRIEDMAN AND S. KAUFMAN, J. Biol. Chem., 241 (1966) 2256. 14 G. LINDSTEDT, Biochemistry, 6 (1967) 1271.
Received April 4th, 1968 Biochim. Biophys. Acta, 158 (1968) 503-505
503
BBA 2 1 2 2 0
~-Ketoglutarate and hydroxylation of ~-butyrobetaine Recently it has been reported that c~-ketoglutarate stimulates four different hydroxylation reactions, i.e. the hydroxylation of peptidyl proline to 4-hydroxyproline 1-3, the hydroxylation of 7-butyrobetaine to carnitine 4,5, the hydroxylation of thymine to 7-hydroxymethyluracil~, and the hydroxylation of peptidyl lysine to 5-hydroxylysineT,8. The same enzyme might hydroxylate both peptidyl proline and peptidyl lysine 7. The hydroxylases have common characteristics, since they all require molecular oxygen, ascorbate and Fe ~+. Pyruvate and oxaloacetate could replace ~-ketoglutarate in the hydroxylation of peptidyl proline, although they were much less effectiveLa. HUTTON,TAPPEL AND UDENFRIEND 3 found no relation between the hydroxylation of peptidyl proline and the disappearance of ~-ketoglutarate, which was ascribed mainly to the presence of transaminases in the crude enzyme preparation. It was suggested by these authors that ~-ketoglutarate might act as an allosteric effector, although a role as a cosubstrate could not be excluded from their experimental data. We have further studied the role of ~-ketoglutarate in the hydroxylation of 7-butyrobetaine by an enzyme from Pseudomonas sp. AK i (ref. 9). The enzyme has been obtained in soluble form by sonication of bacteria which had grown on 7-butyrobetaine as the sole source of carbon and nitrogen and it has been partially purified by chromatography on DEAE-cellulose and on hydroxylapatite. The partially purified enzyme has an absolute requirement for ascorbate, Fe 2+ and for ~-ketoglutarate 5. Several other organic acids have been tested fol their ability to replace ~-ketoglutarate but with negative results. Among others, fumarate, oxaloacetate, pyruvate, ~hydroxyglutarate, ~-keto-y-hydroxyglutarate and c~-ketoadipate have been tested. The change in ~-ketoglutarate concentration during hydroxylation of 7-butyrobetaine has been studied by enzymatic determinations of ~-ketoglutarate with L-glutamate: NAD + oxidoreductase (deaminating) (EC 1.4.1.2 ) obtained from C. F. Boehringer and Soehne GmbH, Mannheim, West Germany. In the assay for ~-ketoglutarate, the disappearance of NADH and also the simultaneous formation of NAD + have been determined by the spectrophotofluorometric methods of KAPLAN,COLOWICK AND BARNES10 and LOWRY, ROBERTS AND KAPPHAHN 11. A stoichiometric relationship has been observed between the formation of carnitine and the disappearance of ~-ketoglutarate in several experiments with varying concentrations of enzyme, ~-ketoglutarate and 7-butyrobetaine. In the presence of high concentrations of enzyme and 7-butyrobetaine, ~-ketoglutarate has been found to be limiting for the yield of hydroxylated product. In the absence of 7-butyrobetaine, there was no change in the concentration of c~-ketoglutarate during incubation with enzyme and other cofactors. Fig. I shows the iesults of an experiment with varying amounts of ~-ketoglutarate and correlates the formation of carnitine with the disappearance of ~-ketoglutarate. To verify the degradation of cc-ketoglutarate, incubations were carried out with E5-1~C~-ketoglutarate obtained from the Radiochemical Centre, Amersham, England. The reaction mixtures were first chromatographed on columns of silicic acid prepared as described by PRIOR I~'ERRAZAND RELVAS12. Fig. 2 shows a typical chromatogram Biochim. Biophys. Acta, 158 (1968) 5o3-5o5
504
PRELIMINARY NOTES
with two peaks containing radioactive material coinciding with ~-ketoglutaric acid and succinic acid, which had been added as carriers immediately before chromatography. The identity of the reaction product with succinic acid has been further established by chromatography on Dowex 2-X8 (2oo-4oo mesh; eluted with o.5 M 1.5
i
i
//
/Ib / / /@
1.C E 3.
@/ /
F
/
E
/ 0
/
/
/
/
1200
24
800
16
/
/
._c E
@// /
/
a 05 "6
2
/
E :3-
E
@// z"
400
I 1 0.5
I 1.0
1.5
Disappearance of 2-ketoglutarate, #moles
%
10
20 Fraction n u m b e r
3o~~ l
Fig. I. R e l a t i o n s h i p between the h y d r o x y l a t i o n of 7 - b u t y r o b e t a i n e to carnitine and the disa p p e a r a n c e of ~-ketoglutarate. The enzyme p r e p a r a t i o n had been prepared by sonication of P s e u d o m o n a s sp. A K I, centrifugation at 75ooo × g, s t r e p t o m y c i n precipitation, filtration t h r o u g h S e p h a d e x G-25, adsorption onto a c o l u m n of h y d r o x y l a p a t i t e and elution of the enzymatic activity with 0.05 M p o t a s s i u m p h o s p h a t e at p H 7.0. The incubation m i x t u r e contained: [rnethyl-t4C]7b u t y r o b e t a i n e , 2 ~moles; a-ketoglutarate, o. 4 #mole; sodium ascorbate, IO/,moles; FeSO 4. 7 H20, o. 4 p mole; catalase, I mg; nicotinamide, 4 ° / t m o l e s ; KC1, 2 5 / , m o l e s ; p o t a s s i u m p h o s p h a t e buffer at p H 7.o, 25/nnoles. The final volume was o. 7 ml. The incubations were carried o u t at 37 ° for 45 nlin. The formation of carnitine was determined by c h r o m a t o g r a p h y on columns of Dowex 5 ° as described in detail previously 14. The disappearance of a-ketoglutarate was determined by enzymatic assay at the end of the incubation period (see text). Fig. 2. C h r o m a t o g r a p h y on a column of silicic acid of the incubation m i x t u r e from enzymatic h y d r o x y l a t i o n of 7 - b u t y r o b e t a i n e to carnitine in the presence of F5-14C]~-ketoglutarate. F o r conditions of i n c u b a t i o n see legend to Fig. i. The c o l u m n was prepared from 8 g of silicic acid (Mallinckrodt, IOO mesh) and eluted with benzene-tert.-butanol (9:1, v/v) in 4-ml fractions. ~-Ketoglutaric acid (IO ing) and succinic acid (I rag) had been added before c h r o m a t o g r a p h y . The isotope c o n t e n t was determined b y counting of dried aliquots in a windowless gas-flow counter, and the r e m a i n d e r of the fractions was titrated with o.o2 M NaOH.
formic acid) and on paper (system 5 of ref. 12). Furthermore the product has been recrystallized to constant specific radioactivity with authentic succinic acid. Thus, the experiments have demonstrated an intimate coupling between the hydroxylation of 7-butyrobetaine to carnitine and the degradation of ~-ketoglutarate. Fumarate activates dopamine/3-hydroxylase, 3,4-dihydloxyphenylethylamine, ascorbate:oxygen oxidoreductase (hydroxylating) (EC I.I4.2.I), which is a copper enzyme requiring ascorbate. FRIEDMAN"AND KAUFMAN13 have suggested that in this case fumarate acts either by combining with the copper ion in a way which facilitates the oxidation of Cu + to Cu 2+ or by changing the conformation of the protein, thereby enchancing the reactivity of Cu +. Apparently, fumarate is not stoichiometrically converted to any other compound during the hydroxylation reaction 1~. Fumarate is not obligatory in the case of dopamine/3-hydroxylase in contrast to ~-ketoglutarate, which is required by peptidyl-proline hydroxylase, 7-butyrobetaine hydroxylase and thymine 7-hydroxylase. It appears unlikely from present data that the effect of Biochim. Biophys. Aria, 158 (1968) 503-505
PRELIMINARY NOTES
505
a-ketoglutarate in these reactions is that of an allosteric effector. One can only speculate upon the possibilities that a-ketoglutarate is responsible for the generation of reducing equivalents or that it is directly involved in the hydroxylation reaction, the degradation occurring as a consequence of the electron shifts during the hydroxylase reaction. This work has been supported by a grant from the Swedish Medical Research Council (I3X-585).
Department of Physiological Chemistry, Chemical Centre, University of Lund, Lund (Sweden)
GORAN LINDSTEDT SVEN LINDSTEDT BIRGIT 0 L A N D E R MARIANNE TOFFT
I J. J. HUTTON, JR., A. L. TAPPEL AND S. UDENFRIEND, Biochem. Biophys. Res. Commun., 2 4 (1966) 179. 2 K. I. KIVlRIKKO AND D. J. PROCKOP, Arch. Biochem. Biophys., 118 (1967) 611. 3 J. J. HUTTON, JR., A. L. TAPPEL AND S. UDENFRIEND, Arch. Biochem. Biophys., 118 (1967) 231. 4 G. LINOSTEDT, Dissertation, Karolinska I n s t i t u t e t , Stockholm, 1967. 5 G. LINDSTEDT, S. LINDSTEDT, T. MIDTVEDT AND M. TOFFT, Bioehem. J., lO 3 (1967) i9P. 6 M. T. ABBOTT, E. K. SCHANDL, R. F. LEE, T. S. PARKER AND R. J. MIDGETT, Biochim. Biophys. Acta, 132 (1967) 525 . 7 K. I. KIVIRIKKO AND D. J. PROCKOP, Proc. Natl. Acad. Sci. U.S., 57 (1967) 782. 8 E. HAUSMANN, Biochim. Biophys. Acta, 133 (1967) 591. 9 G. LINDSTEDT, S. LINDSTEDT, T. MIDTVEDT AND M. TOFFT, Biochemistry, 6 (1967) 1262. IO N. O. KAPLAN, S. P. COLOWICK AND C. C. BARNES, J. Biol. Chem., 191 (1951) 461. I I O. H. LOWRY, N. R. ROBERTS AND J. I. KAPPHAHN, J. Biol. Chem., 224 (1957) lO47. 12 F. G. PRIOR FERRAZ AND M. E. RELVAS, Clin. Chim. Acta, i i (1965) 244. 13 S. FRIEDMAN AND S. KAUFMAN, J. Biol. Chem., 241 (1966) 2256. 14 G. LINDSTEDT, Biochemistry, 6 (1967) 1271.
Received April 4th, 1968 Biochim. Biophys. Acta, 158 (1968) 503-505