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Some considerations concerning the nature of the enzymic galactose-glucose conversion
588
PRELIMINARY NOTES
VOL. 2 2
(I956)
Some considerations concerning the nature of the enzymic galactose-glucose conversion I n v e r s i o n of t h e 4 - h y d r o x y l in t h e conversion of galactose to glucose h a s been t h e s u b j e c t of n u m e r o u s studies. T h e m o s t significant progress in later y e a r s w a s m a d e b y LELOIR1 w h o s h o w e d t h a t t h e e n z y m i c inversion does n o t t a k e place if free galactose or g a l a c t o s e - I - p h o s p h a t e are a d d e d to dialyzed e x t r a c t s of g a l a c t o s e - a d a p t e d yeast. T h e c o m p o u n d s u n d e r g o i n g t h e i n v e r s i o n are uridine d i p h o s p h o g a l a c t o s e (UDPGal) a n d uridine d i p h o s p h o g l u c o s e (UDPG) 1. T h e e n z y m e c a t a l y z i n g t h e e q u i l i b r i u m U D P G a l ~ U D P G is called "galacto-waldenase"*. I n o u r l a b o r a t o r y " g a l a c t o - w a l d e n a s e " from calf liver h a s been purified to a considerable e x t e n t s a n d it h a s been f o u n d t h a t s u c h purified p r e p a r a t i o n s h a v e a n absolute r e q u i r e m e n t for d i p h o s p h o p y r i d i n e nucleotides (DPN) 7. W e h a v e been interested in s t u d y i n g t h e possible e n z y m i c i n t e r c h a n g e of t h e 4 or 5 c a r b o n b o u n d h y d r o g e n of t h e h e x o s e p a r t with h y d r o g e n ions f r o m water, c a t a l y z e d b y this e n z y m e . Kinetic studies in 96 to 9 7 % d e u t e r i u m w a t e r (D20) s h o w e d t h a t t h e r a t e of t h e " g a l a c t o w a l d e n a s e " - c a t a l y z e d c o n v e r s i o n in D 2 0 was n o t to a n y d e t e c t a b l e degree different from t h a t in H~O (see Table I). If, therefore, a d e u t e r i u m e x c h a n g e took place, this process could n o t be t h e limiting factor. TABLE I RATES OF "GAL-WALDENASE" IN 8 2 0
AND D 2 0
R e a c t i o n m i x t u r e s in 95 % D.20 or in H 2 0 as indicated, c o n t a i n e d o.o33/*mole of U D P G a l , o.I # m o l e of D P N , a n d 7-5/~g of purified Gal-waldenase in a t o t a l v o l u m e of 425/~1 of o.oSM glycine p H 8.9. R e a c t i o n was s t o p p e d b y h e a t i n g for 3 ° seconds at IOO °, a n d 4oo/~1 s a m p l e s were a s s a y e d for U D P G f o r m e d with U D P G d e h y d r o g e n a s e 11. Time o~ incubation (minutes)
5 IO
UDPG /ormed (l~moles) § in D,20
O"O15 O.O2I
in H20
O"O17 O.O23
§ Owing to U D P G originally c o n t a m i n a t i n g t h e U D P G a l p r e p a r a t i o n a b l a n k of o.oo 5 h a s been subtracted. If purified "galacto-waldenase" is m a d e to o p e r a t e on U D P G a l in t r i t i u m - e n r i c h e d w a t e r (about o.oooi % enriched w i t h T~O 2.5 mc/ml), t h e following c h a n g e s were o b s e r v e d (Table II). A p p a r e n t l y no t r i t i u m u p t a k e t o o k place a l t h o u g h t h e e n z y m e c a t a l y z e d t h e conversion of considerable a m o u n t s of U D P G a l to U D P G . T h i s lack of e x c h a n g e w i t h w a t e r is p r o b a b l y n o t d u e to a d i s c r i m i n a t i o n a g a i n s t T~O. Otherwise, t h e reaction rate in 96 % D 2 0 w o u l d h a v e been g r e a t l y reduced as c o m p a r e d w i t h t h e r a t e in H 2 0 . I t h a s so far n o t been possible to d e t e c t a n y t r i t i u m in t h e u r i d i n e nucleotide w h e n t h e reaction was carried o u t in t h e presence of D P N H or D P N , b o t h labeled w i t h t r i t i u m in t h e 4-position of t h e n i c o t i n a m i d e ring (E. MAXWELL, u n p u b l i s h e d studies). E n z y m i c d e h y d r o g e n a t i o n of deutero-labelled D - C - O H g r o u p s to t h e c o r r e s p o n d i n g c a r b o n y l groups w i t h D P N as D t r a n s f e r s y s t e m s h o w s a lack of e x c h a n g e of c a r b o n - b o u n d d e u t e r i u m s with t h e p r o t o n s of water. T h e " g a l a c t o - w a l d e n a s e " - c a t a l y z e d reaction, in w h i c h D P N is likewise * It is still c o m m o n to s t a t e t h a t t h e conversion of a - g a l a c t o s e - i - p h o s p h a t e to a-glucose1 - p h o s p h a t e is c a t a l y z e d b y " g a l a c t o - w a l d e n a s e " a n d t h a t U D P G is t h e c o e n z y m e (see, for nstance, I n t e r m e d i a r y M e t a b o l i s m ChartS). T h i s a m b i g u o u s t e r m i n o l o g y s e e m s q u i t e u n n e c e s s a r y ; t h e p r o b l e m s were clarified as early as 19513 , a n d t h e first step e n z y m e w h i c h c a t a l y z e s t h e i n c o r p o r a t i o n of a - g a l a c t o s e - i - p h o s p h a t e (Gal-I-P) into t h e uridine nucleotide was f o u n d a n d described in 1953 a n d 19544,5. P e r h a p s t h e t e r m "waldenase" s h o u l d be a b a n d o n e d altogether. T h e results described in t h e p r e s e n t article t h r o w f u r t h e r d o u b t on t h e h y p o t h e s i s t h a t a W a l d e n inversion is involved in this reaction. W e w o u l d therefore r e c o m m e n d t h e n a m e U D P G a l 4e p i m e r a s e as a relatively descriptive a n d correct n a m e for t h e e n z y m e w h i c h c a t a l y z e s t h e i n v e r s i o n step. T h e n a m e " g a l - i - P uridyl t r a n s f e r a s e " (abbrev.: P G a l - U - T r a n s f e r a s e ) w o u l d be logical for t h e e n z y m e w h i c h c a t a l y z e s t h e incorporation of G a l - I - P into U D P G , a step in w h i c h a - g a l a c t o s e - i - p h o s p h a t e acts as a nucleophilic r e a g e n t for t h e uridyl m o i e t y of U D P G (c[. 5). T h e biological implications of t h e resolutions of t h e s e two step e n z y m e s will n o t be discussed here.
VOL. 2 2
(1956)
589
PRELIMINARY NOTES T A B L E II LACK
OF
INCORPORATION
OF
TRITIUM
INTO
UDPG
Reaction m i x t u r e consisted of 1.15 ml tritium-enriched w a t e r (2.5 mc/ml), 2o/zl 3 M glycine p H 8.9, 75/~1 U D P G a l (6.6;umoles/ml), io #1 D P N (50 pmoles/ml) and 30 pl purified Galwaldenase (2.5 mg protein/ml). Controls consisted of (i) the s a m e m i x t u r e w i t h o u t D P N and (2) the same m i x t u r e with heat-inactivated waldenase I n c u b a t i o n was for 6 h o u r s at r o o m t e m p e r a t u r e . Reaction was s t o p p e d by heating for 3 ° sec at too% A 3 ° / z l aliquot was assayed for U D P G formation using U D P G dehydrogenase. Nucleotides were adsorbed on Darco, washed repeatedly with o.ooi N HC1 to remove unreacted tritium, and eluted with o . o 2 M cyclohexylamine in 5 ° % ethanol. An aliquot was taken to determine U D P G recovered and the remaining eluates were evaporated to o.I ml and counted in a scintillation counter. Reaction mixture
UDPG /ormed
UDPG counted
c.p.m.
I) 2) 3) 4)
0.39 none none --
0.32 ----
7° 50 75 825"
complete without DPN with heated waldenase + 20/~1 t r i t i u m w a t e r diluted i : IO,OOO
* Based on a counting efficiency of 7 % as indicated b y the internal standard, the incorporation of I /,mole H f r o m m e d i u m per /~mole U D P G formed would give i i o o c.p.m, in sample No. i. involved, bears p r o b a b l y a close relationship to the a b o v e - m e n t i o n e d t y p e s of reactions ; the lack of u p t a k e of labeled h y d r o g e n from w a t e r agrees with this contention. KAUEMAN, KORKES AND DEL CAMPILLO 9 have found t h a t lactate racemase requires D P N and recently such a r e q u i r e m e n t has also been found for f l - h y d r o x y b u t y r y l CoA racemase TM. This altogether makes it worthwhile to consider the possibility t h a t "galacto-waldenase" catalyzes a t y p e of epimerization reaction. The i n t e r m e d i a r y m a y be a 4-keto s u g a r or a h y d r a t e d 4-keto sugar. I n the latter case the e n z y m e catalyzing the inversion should, as suggested in the footnote on p. 588, be called U D P - h e x o s e - 4 - h y d r o x y l epimerase (for terminology see ref. is).
Acknowledgement. We are grateful to Drs. DEWITT STETTEN AND Y. TOPPER for samples of pure deuterium w a t e r and to Dr. R. PETERSON and Miss MARGARET BOLLIER for help in determining t r i t i u m in the scintillation counter. HERMAN M. KALCKAR National Institute o/Arthritis and Metabolic Diseases, ELIZABETH S. MAXWELL
National Institutes of Health, Bethesda, Maryland (U.S.A.) 1 L. F. LELOIR, in W. D. MCELROY AND B. GLASS, Phosphorus Metabolism, Vol. I, J o h n s H o p k i n s Press, Baltimore, 1951, p. 67. 2 Intermediary Metabolism (Copyright 1955, b y H. J. SALLACH AND R. W. McGILVERY, GME, Madison, Wisconsin). s L. F. LELOIR, Arch. Biochem. Biophys., 33 (1951) 186. 4 H . M. KALCKAR, B. BRAGANCA AND A. MUNCH-PETERSEN, Nature, 172 (1953) lO39. 5 H. M. KALCKAR AND H. KLENOW, Ann. Rev. Biochem., 23 (1954) 527, see especially p. 546. e E . S. MAXWELL, u n p u b l i s h e d data. E. S. MAXWELL, J. Am. Chem. See., 78 (1956) lO74. 8 B . VENNESLAND AND F. H. WESTHEIMER, in W. D. MCELROY AND B . GLASS, The Mechanism o~ Enzyme Action, J o h n s H o p k i n s Press, Baltimore, 1953, p. 357. 9 S. KAUFMAN, S. KORKES AND A. DEL CAMPILLO, J . Biol. Chem., 192 (I951) 3Ol. 10 S. J . WAKIL, Biochim. Biophys. Acta, 18 (1955) 314 . 11 j . L. STROMINGER, H. M. KALCKAR AND E. S. MAXWELL, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. III, Academic Press, Inc., New York, (in press). 12 p . K . STUMPF AND B . L. HORECKER, J . Biol. Chem., 218 (1956) 753Received S e p t e m b e r 6th, 1956
Addendum Drs. L. ANDERSON, A. M. LANDEL AND D . F . DIEDRICH, a s well as Drs. A. KOWALSKY AND D . E . KOSHLAND h a v e recently informed us t h a t in the galactose-glucose conversion b y L. bulgaricus neither 1sO nor t r i t i u m are t a k e n up f r o m w a t e r enriched with one or the other isotope. The latter g r o u p has also furnished evidence showing t h a t D P N is b o u n d to the bacterial galacto-waldenase and also here it seems to be required for the reaction.
PRELIMINARY NOTES
VOL. 2 2
(I956)
Some considerations concerning the nature of the enzymic galactose-glucose conversion I n v e r s i o n of t h e 4 - h y d r o x y l in t h e conversion of galactose to glucose h a s been t h e s u b j e c t of n u m e r o u s studies. T h e m o s t significant progress in later y e a r s w a s m a d e b y LELOIR1 w h o s h o w e d t h a t t h e e n z y m i c inversion does n o t t a k e place if free galactose or g a l a c t o s e - I - p h o s p h a t e are a d d e d to dialyzed e x t r a c t s of g a l a c t o s e - a d a p t e d yeast. T h e c o m p o u n d s u n d e r g o i n g t h e i n v e r s i o n are uridine d i p h o s p h o g a l a c t o s e (UDPGal) a n d uridine d i p h o s p h o g l u c o s e (UDPG) 1. T h e e n z y m e c a t a l y z i n g t h e e q u i l i b r i u m U D P G a l ~ U D P G is called "galacto-waldenase"*. I n o u r l a b o r a t o r y " g a l a c t o - w a l d e n a s e " from calf liver h a s been purified to a considerable e x t e n t s a n d it h a s been f o u n d t h a t s u c h purified p r e p a r a t i o n s h a v e a n absolute r e q u i r e m e n t for d i p h o s p h o p y r i d i n e nucleotides (DPN) 7. W e h a v e been interested in s t u d y i n g t h e possible e n z y m i c i n t e r c h a n g e of t h e 4 or 5 c a r b o n b o u n d h y d r o g e n of t h e h e x o s e p a r t with h y d r o g e n ions f r o m water, c a t a l y z e d b y this e n z y m e . Kinetic studies in 96 to 9 7 % d e u t e r i u m w a t e r (D20) s h o w e d t h a t t h e r a t e of t h e " g a l a c t o w a l d e n a s e " - c a t a l y z e d c o n v e r s i o n in D 2 0 was n o t to a n y d e t e c t a b l e degree different from t h a t in H~O (see Table I). If, therefore, a d e u t e r i u m e x c h a n g e took place, this process could n o t be t h e limiting factor. TABLE I RATES OF "GAL-WALDENASE" IN 8 2 0
AND D 2 0
R e a c t i o n m i x t u r e s in 95 % D.20 or in H 2 0 as indicated, c o n t a i n e d o.o33/*mole of U D P G a l , o.I # m o l e of D P N , a n d 7-5/~g of purified Gal-waldenase in a t o t a l v o l u m e of 425/~1 of o.oSM glycine p H 8.9. R e a c t i o n was s t o p p e d b y h e a t i n g for 3 ° seconds at IOO °, a n d 4oo/~1 s a m p l e s were a s s a y e d for U D P G f o r m e d with U D P G d e h y d r o g e n a s e 11. Time o~ incubation (minutes)
5 IO
UDPG /ormed (l~moles) § in D,20
O"O15 O.O2I
in H20
O"O17 O.O23
§ Owing to U D P G originally c o n t a m i n a t i n g t h e U D P G a l p r e p a r a t i o n a b l a n k of o.oo 5 h a s been subtracted. If purified "galacto-waldenase" is m a d e to o p e r a t e on U D P G a l in t r i t i u m - e n r i c h e d w a t e r (about o.oooi % enriched w i t h T~O 2.5 mc/ml), t h e following c h a n g e s were o b s e r v e d (Table II). A p p a r e n t l y no t r i t i u m u p t a k e t o o k place a l t h o u g h t h e e n z y m e c a t a l y z e d t h e conversion of considerable a m o u n t s of U D P G a l to U D P G . T h i s lack of e x c h a n g e w i t h w a t e r is p r o b a b l y n o t d u e to a d i s c r i m i n a t i o n a g a i n s t T~O. Otherwise, t h e reaction rate in 96 % D 2 0 w o u l d h a v e been g r e a t l y reduced as c o m p a r e d w i t h t h e r a t e in H 2 0 . I t h a s so far n o t been possible to d e t e c t a n y t r i t i u m in t h e u r i d i n e nucleotide w h e n t h e reaction was carried o u t in t h e presence of D P N H or D P N , b o t h labeled w i t h t r i t i u m in t h e 4-position of t h e n i c o t i n a m i d e ring (E. MAXWELL, u n p u b l i s h e d studies). E n z y m i c d e h y d r o g e n a t i o n of deutero-labelled D - C - O H g r o u p s to t h e c o r r e s p o n d i n g c a r b o n y l groups w i t h D P N as D t r a n s f e r s y s t e m s h o w s a lack of e x c h a n g e of c a r b o n - b o u n d d e u t e r i u m s with t h e p r o t o n s of water. T h e " g a l a c t o - w a l d e n a s e " - c a t a l y z e d reaction, in w h i c h D P N is likewise * It is still c o m m o n to s t a t e t h a t t h e conversion of a - g a l a c t o s e - i - p h o s p h a t e to a-glucose1 - p h o s p h a t e is c a t a l y z e d b y " g a l a c t o - w a l d e n a s e " a n d t h a t U D P G is t h e c o e n z y m e (see, for nstance, I n t e r m e d i a r y M e t a b o l i s m ChartS). T h i s a m b i g u o u s t e r m i n o l o g y s e e m s q u i t e u n n e c e s s a r y ; t h e p r o b l e m s were clarified as early as 19513 , a n d t h e first step e n z y m e w h i c h c a t a l y z e s t h e i n c o r p o r a t i o n of a - g a l a c t o s e - i - p h o s p h a t e (Gal-I-P) into t h e uridine nucleotide was f o u n d a n d described in 1953 a n d 19544,5. P e r h a p s t h e t e r m "waldenase" s h o u l d be a b a n d o n e d altogether. T h e results described in t h e p r e s e n t article t h r o w f u r t h e r d o u b t on t h e h y p o t h e s i s t h a t a W a l d e n inversion is involved in this reaction. W e w o u l d therefore r e c o m m e n d t h e n a m e U D P G a l 4e p i m e r a s e as a relatively descriptive a n d correct n a m e for t h e e n z y m e w h i c h c a t a l y z e s t h e i n v e r s i o n step. T h e n a m e " g a l - i - P uridyl t r a n s f e r a s e " (abbrev.: P G a l - U - T r a n s f e r a s e ) w o u l d be logical for t h e e n z y m e w h i c h c a t a l y z e s t h e incorporation of G a l - I - P into U D P G , a step in w h i c h a - g a l a c t o s e - i - p h o s p h a t e acts as a nucleophilic r e a g e n t for t h e uridyl m o i e t y of U D P G (c[. 5). T h e biological implications of t h e resolutions of t h e s e two step e n z y m e s will n o t be discussed here.
VOL. 2 2
(1956)
589
PRELIMINARY NOTES T A B L E II LACK
OF
INCORPORATION
OF
TRITIUM
INTO
UDPG
Reaction m i x t u r e consisted of 1.15 ml tritium-enriched w a t e r (2.5 mc/ml), 2o/zl 3 M glycine p H 8.9, 75/~1 U D P G a l (6.6;umoles/ml), io #1 D P N (50 pmoles/ml) and 30 pl purified Galwaldenase (2.5 mg protein/ml). Controls consisted of (i) the s a m e m i x t u r e w i t h o u t D P N and (2) the same m i x t u r e with heat-inactivated waldenase I n c u b a t i o n was for 6 h o u r s at r o o m t e m p e r a t u r e . Reaction was s t o p p e d by heating for 3 ° sec at too% A 3 ° / z l aliquot was assayed for U D P G formation using U D P G dehydrogenase. Nucleotides were adsorbed on Darco, washed repeatedly with o.ooi N HC1 to remove unreacted tritium, and eluted with o . o 2 M cyclohexylamine in 5 ° % ethanol. An aliquot was taken to determine U D P G recovered and the remaining eluates were evaporated to o.I ml and counted in a scintillation counter. Reaction mixture
UDPG /ormed
UDPG counted
c.p.m.
I) 2) 3) 4)
0.39 none none --
0.32 ----
7° 50 75 825"
complete without DPN with heated waldenase + 20/~1 t r i t i u m w a t e r diluted i : IO,OOO
* Based on a counting efficiency of 7 % as indicated b y the internal standard, the incorporation of I /,mole H f r o m m e d i u m per /~mole U D P G formed would give i i o o c.p.m, in sample No. i. involved, bears p r o b a b l y a close relationship to the a b o v e - m e n t i o n e d t y p e s of reactions ; the lack of u p t a k e of labeled h y d r o g e n from w a t e r agrees with this contention. KAUEMAN, KORKES AND DEL CAMPILLO 9 have found t h a t lactate racemase requires D P N and recently such a r e q u i r e m e n t has also been found for f l - h y d r o x y b u t y r y l CoA racemase TM. This altogether makes it worthwhile to consider the possibility t h a t "galacto-waldenase" catalyzes a t y p e of epimerization reaction. The i n t e r m e d i a r y m a y be a 4-keto s u g a r or a h y d r a t e d 4-keto sugar. I n the latter case the e n z y m e catalyzing the inversion should, as suggested in the footnote on p. 588, be called U D P - h e x o s e - 4 - h y d r o x y l epimerase (for terminology see ref. is).
Acknowledgement. We are grateful to Drs. DEWITT STETTEN AND Y. TOPPER for samples of pure deuterium w a t e r and to Dr. R. PETERSON and Miss MARGARET BOLLIER for help in determining t r i t i u m in the scintillation counter. HERMAN M. KALCKAR National Institute o/Arthritis and Metabolic Diseases, ELIZABETH S. MAXWELL
National Institutes of Health, Bethesda, Maryland (U.S.A.) 1 L. F. LELOIR, in W. D. MCELROY AND B. GLASS, Phosphorus Metabolism, Vol. I, J o h n s H o p k i n s Press, Baltimore, 1951, p. 67. 2 Intermediary Metabolism (Copyright 1955, b y H. J. SALLACH AND R. W. McGILVERY, GME, Madison, Wisconsin). s L. F. LELOIR, Arch. Biochem. Biophys., 33 (1951) 186. 4 H . M. KALCKAR, B. BRAGANCA AND A. MUNCH-PETERSEN, Nature, 172 (1953) lO39. 5 H. M. KALCKAR AND H. KLENOW, Ann. Rev. Biochem., 23 (1954) 527, see especially p. 546. e E . S. MAXWELL, u n p u b l i s h e d data. E. S. MAXWELL, J. Am. Chem. See., 78 (1956) lO74. 8 B . VENNESLAND AND F. H. WESTHEIMER, in W. D. MCELROY AND B . GLASS, The Mechanism o~ Enzyme Action, J o h n s H o p k i n s Press, Baltimore, 1953, p. 357. 9 S. KAUFMAN, S. KORKES AND A. DEL CAMPILLO, J . Biol. Chem., 192 (I951) 3Ol. 10 S. J . WAKIL, Biochim. Biophys. Acta, 18 (1955) 314 . 11 j . L. STROMINGER, H. M. KALCKAR AND E. S. MAXWELL, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. III, Academic Press, Inc., New York, (in press). 12 p . K . STUMPF AND B . L. HORECKER, J . Biol. Chem., 218 (1956) 753Received S e p t e m b e r 6th, 1956
Addendum Drs. L. ANDERSON, A. M. LANDEL AND D . F . DIEDRICH, a s well as Drs. A. KOWALSKY AND D . E . KOSHLAND h a v e recently informed us t h a t in the galactose-glucose conversion b y L. bulgaricus neither 1sO nor t r i t i u m are t a k e n up f r o m w a t e r enriched with one or the other isotope. The latter g r o u p has also furnished evidence showing t h a t D P N is b o u n d to the bacterial galacto-waldenase and also here it seems to be required for the reaction.