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Phospho-2-keto-d -gluconate, an intermediate in the carbohydrate metabolism of aerobacter cloacae
3LUCONATE,
AN
INTERMEDI
ETABOLISM
OF AEROBACTE1
?
hv J. DlL 1A;V
Veterinary College, Stale University, (;t~e )ed t h e presence of a n e w a d a p t i v e enz, ae. This e n z y m e c a t a l y z e s t h e t r a n s p h ce of Mg ions. \Ve n o w s uc c e e de d in i t i o n : a p h o s p h o r i c e s t e r of 2-ketoglucc an i n t e r m e d i a t e in t h e h e x o s e - m o n o p h
aally called from ATI ~d i s o l a t i n g pho-2-ketot t i v e route,
t~eu-iree e n z y m e p r e p a r a t i o n s of oi 22-KetogluconoKinase wen prepared - k e t o g l u c o n o k i n a s e were xl b a c t e r i a : d i s r u p t i n g t h e m w i t h glass p o w d e r s or a l u m i n a 7 a t 4 :~C, or a f t e r crushi G-HES press ~ alumina at 2o ° C. B e s t r e s u l t s were o b t a i n e d w i t h t h e a l u nm i n a m e t h o GHES press. p h o s p h o r y l a t i o n of 2 - k e t o g l u c o n a t e w i t h AT I' w a s s t udi u d ie d w i t h t h e m e t h o d .of )WICK AND KALCKAR1, b y p a p e r c h r o m a t o g r a p h y a n d w i t h s e v e r a l col< t a pe r ehro ) g r a p h y r e v e a l e d tile presence of a r e d u c i n g s u b s t a n c e w whic hich gave a " violet color o - p h e n y l e n e d i a m i n e - H C 1 s p r a y L To follow t h e e n z y m e re reaa c t i on, we ~'cific p a p e r m a t o g r a p h i c m e t h o d , w h i c h a l l o w e d the s e p a r a t e d e t e r m i n ai t i o n of t h ad t h e ne w •m e d i a t e (see Fig. J). This u n k n o w n s u b s t a n c e w a s n o t i d e n t i c a l w i t h phosphate : or r e d u c i n g s u b s t a n c e . I t w a s s h o w n to be a p h o s p h o r i c ester. Thi s ts p r e p a r e d ,rge scale e x p e r i m e n t s a n d precipitatei1 in t h e fraction of th, the barium s ~1 i ns ol uhh' p h o r i c esters. H y d r o l y s i s w i t h a l k a l i n e p h o s p h a t a s e r e s u l t e d in t he f eo r nm a t i o n of i sphate and t o g l u c o n a t e . The q u i n o x a l i n e d e r i v a t e of t h e new s u b s t a n c e g a v e tl absorption t r m n as 2 - k e t o g l u c o n o q u i n o x a l i n e . C h r o m a t o g r a p h i c e v i d e n c e w a s also a phospho t o g l u c o n a t e . I t is m o s t p r o b a b l y t h e 6 - p h o s p)hhoe - d e r i v a t i v e . O ur p m ons so far a i n e d a b o u t 8o % of the new c o m p o u n d . E n z y m e p r e p) a r a t i o n s , o b t a ~ss powder, m p o s e t h i s s u b s t a n c e f u r t h e r a n d 3 - p h o s p h o - D - g l y c e r a t e is formed. fc F u r t h e r w o r k on its isolation, purification, a n a l y s i s a n d m e t a b o l i s m is in progress. I t is he],ped ublisb a full a c c o u n t of t h i s w o r k in t h e n e a r future. The aa~u t h o r is i n d e b t e d t o t he N a t i o rn a a l Fern ~ts v e e r \ V e t e n s c h a p p e l i j k O n d e r z o e k for a grant. Fig. ~. F o r m a t i o n of l ' h o s p~ho-2-keto-D-gluconate ho-: from 2-keto --ket~ l>Extinction at 350 m p g l u c o n a t e . B a c t e r i a , c u l t u r e d o n 2 - k e t o g l u c o n a t e m e d i u m , v~'er(" ~ h a r v e s t e d , w a s h e d a n d c r u s h e d w i t h e q u a l w e i g h t of a l u m i n a in t h e (14 HUGHES press a t -2o ° C. E x t r a c t i o n w i t h o.o 5 3 I Tris buffer p t f 7.4. 2 - K e t o g l u c o n o k i n a s e w a s p r e c i p i t a t e d in t he f r a c t i o n o.3 s a t u r a t e d a m m o n i u m sulfate. Co C o m p o s i t i o n of t h e r e a c t i o n m i x t ut r e : 0.3 a - A T I '. o.2 ml e n z y m e solution, 1 6 / ,~M , M Mg++, x'v 2 o / * M N a F , 17 ~tell N Na-A" ohlnle to # 3 f N a - 2 - k e t o g l u e o n a t e , o.o 5 M Tris buffer p H 7.4,final vvolu - inl. Temp. 3 ° C. At t h e t i m e i n d i c a t e d in tim gra ph, 6 o / , I x~ were 0.2 R - I 2 o in t h e H+ form a n d s p) o t t e d on d e c a t i o n i z e d w i t h A i n b e r l i t e lIRp r e w a s h e d \ V h a t m a n No. J. The c h r o m a t o g r a m w a s r u n ove rni g h t l l ow e d at 4 C u s i n g 0 m e t h a n o l / I a m m o n i a / 3 w a t e r as s ol ve nt . This aallox a clean s e p a r a t i o n of t h e c o m p o n e n t s . After s p r a y i n g w i t h o-p~be0.1 n y l e n e d i a m i n e - H C 1a a n d h e a t i n g a t 95 ° C for 2.5 m i n u t e s , 2-ket~, 2-M g l u c o n a t e shows up as a c l e a r gre e ni s h s pot (RF o.63) a n d t he nex~ r p h o s p h a t e e s t e r as a v i o l e t s p o t (RF o.36). The colored s pot s are c u t O hour's out, e l u t e d w i t h m e t h a n o l a n d t he e x t i n c t i o n of t h e q u i n o x a l i n e s ormati on. d e t e r m i n e d a t 35 o nv*. Curve 2K : 2 - k e t o g l u c o n a t e decrease ; 2 K P : p h o s p h o - 2 - k e t o g l u c o n a t e fforma RI c ["1,2R E N C E S P. COLOWICK AND H. M. KALCKAR :ALCKAR, ./. Biol. Che*n., I 4 8 ([943) I17DE LEY, Enzymologia, i 6 (1953) 99. DmKENS, Biochem. J., 3z () 9 3 8 ) 16"6. E. H~JanES, Brit. J. Exp. Path., 32 (t95 I) 975 5 I . C. L A N N I N G AND S. S. C O H~EN, E N J. Biol. Chem.. I89 ( t 9 5 Q 1o9. {i F. L I P M A N N , Nature, i38 (i936) 588. 7 H. Me ILLWMN, J. Gen. Microb. 9b., 2 (1948) 288. AN, M. l". ITTER AND ( . tq. ~VERKMAN, lotva Slate Coll..]. £'ci. ~4 8 ~V. P . ~¢VIGGERT, M. SILVERMAN 0 9 4 o) I79. R e c e i v e d D e c e m b e r -'9tlI, ~953 i S. J. a F. D.
AN
INTERMEDI
ETABOLISM
OF AEROBACTE1
?
hv J. DlL 1A;V
Veterinary College, Stale University, (;t~e )ed t h e presence of a n e w a d a p t i v e enz, ae. This e n z y m e c a t a l y z e s t h e t r a n s p h ce of Mg ions. \Ve n o w s uc c e e de d in i t i o n : a p h o s p h o r i c e s t e r of 2-ketoglucc an i n t e r m e d i a t e in t h e h e x o s e - m o n o p h
aally called from ATI ~d i s o l a t i n g pho-2-ketot t i v e route,
t~eu-iree e n z y m e p r e p a r a t i o n s of oi 22-KetogluconoKinase wen prepared - k e t o g l u c o n o k i n a s e were xl b a c t e r i a : d i s r u p t i n g t h e m w i t h glass p o w d e r s or a l u m i n a 7 a t 4 :~C, or a f t e r crushi G-HES press ~ alumina at 2o ° C. B e s t r e s u l t s were o b t a i n e d w i t h t h e a l u nm i n a m e t h o GHES press. p h o s p h o r y l a t i o n of 2 - k e t o g l u c o n a t e w i t h AT I' w a s s t udi u d ie d w i t h t h e m e t h o d .of )WICK AND KALCKAR1, b y p a p e r c h r o m a t o g r a p h y a n d w i t h s e v e r a l col< t a pe r ehro ) g r a p h y r e v e a l e d tile presence of a r e d u c i n g s u b s t a n c e w whic hich gave a " violet color o - p h e n y l e n e d i a m i n e - H C 1 s p r a y L To follow t h e e n z y m e re reaa c t i on, we ~'cific p a p e r m a t o g r a p h i c m e t h o d , w h i c h a l l o w e d the s e p a r a t e d e t e r m i n ai t i o n of t h ad t h e ne w •m e d i a t e (see Fig. J). This u n k n o w n s u b s t a n c e w a s n o t i d e n t i c a l w i t h phosphate : or r e d u c i n g s u b s t a n c e . I t w a s s h o w n to be a p h o s p h o r i c ester. Thi s ts p r e p a r e d ,rge scale e x p e r i m e n t s a n d precipitatei1 in t h e fraction of th, the barium s ~1 i ns ol uhh' p h o r i c esters. H y d r o l y s i s w i t h a l k a l i n e p h o s p h a t a s e r e s u l t e d in t he f eo r nm a t i o n of i sphate and t o g l u c o n a t e . The q u i n o x a l i n e d e r i v a t e of t h e new s u b s t a n c e g a v e tl absorption t r m n as 2 - k e t o g l u c o n o q u i n o x a l i n e . C h r o m a t o g r a p h i c e v i d e n c e w a s also a phospho t o g l u c o n a t e . I t is m o s t p r o b a b l y t h e 6 - p h o s p)hhoe - d e r i v a t i v e . O ur p m ons so far a i n e d a b o u t 8o % of the new c o m p o u n d . E n z y m e p r e p) a r a t i o n s , o b t a ~ss powder, m p o s e t h i s s u b s t a n c e f u r t h e r a n d 3 - p h o s p h o - D - g l y c e r a t e is formed. fc F u r t h e r w o r k on its isolation, purification, a n a l y s i s a n d m e t a b o l i s m is in progress. I t is he],ped ublisb a full a c c o u n t of t h i s w o r k in t h e n e a r future. The aa~u t h o r is i n d e b t e d t o t he N a t i o rn a a l Fern ~ts v e e r \ V e t e n s c h a p p e l i j k O n d e r z o e k for a grant. Fig. ~. F o r m a t i o n of l ' h o s p~ho-2-keto-D-gluconate ho-: from 2-keto --ket~ l>Extinction at 350 m p g l u c o n a t e . B a c t e r i a , c u l t u r e d o n 2 - k e t o g l u c o n a t e m e d i u m , v~'er(" ~ h a r v e s t e d , w a s h e d a n d c r u s h e d w i t h e q u a l w e i g h t of a l u m i n a in t h e (14 HUGHES press a t -2o ° C. E x t r a c t i o n w i t h o.o 5 3 I Tris buffer p t f 7.4. 2 - K e t o g l u c o n o k i n a s e w a s p r e c i p i t a t e d in t he f r a c t i o n o.3 s a t u r a t e d a m m o n i u m sulfate. Co C o m p o s i t i o n of t h e r e a c t i o n m i x t ut r e : 0.3 a - A T I '. o.2 ml e n z y m e solution, 1 6 / ,~M , M Mg++, x'v 2 o / * M N a F , 17 ~tell N Na-A" ohlnle to # 3 f N a - 2 - k e t o g l u e o n a t e , o.o 5 M Tris buffer p H 7.4,final vvolu - inl. Temp. 3 ° C. At t h e t i m e i n d i c a t e d in tim gra ph, 6 o / , I x~ were 0.2 R - I 2 o in t h e H+ form a n d s p) o t t e d on d e c a t i o n i z e d w i t h A i n b e r l i t e lIRp r e w a s h e d \ V h a t m a n No. J. The c h r o m a t o g r a m w a s r u n ove rni g h t l l ow e d at 4 C u s i n g 0 m e t h a n o l / I a m m o n i a / 3 w a t e r as s ol ve nt . This aallox a clean s e p a r a t i o n of t h e c o m p o n e n t s . After s p r a y i n g w i t h o-p~be0.1 n y l e n e d i a m i n e - H C 1a a n d h e a t i n g a t 95 ° C for 2.5 m i n u t e s , 2-ket~, 2-M g l u c o n a t e shows up as a c l e a r gre e ni s h s pot (RF o.63) a n d t he nex~ r p h o s p h a t e e s t e r as a v i o l e t s p o t (RF o.36). The colored s pot s are c u t O hour's out, e l u t e d w i t h m e t h a n o l a n d t he e x t i n c t i o n of t h e q u i n o x a l i n e s ormati on. d e t e r m i n e d a t 35 o nv*. Curve 2K : 2 - k e t o g l u c o n a t e decrease ; 2 K P : p h o s p h o - 2 - k e t o g l u c o n a t e fforma RI c ["1,2R E N C E S P. COLOWICK AND H. M. KALCKAR :ALCKAR, ./. Biol. Che*n., I 4 8 ([943) I17DE LEY, Enzymologia, i 6 (1953) 99. DmKENS, Biochem. J., 3z () 9 3 8 ) 16"6. E. H~JanES, Brit. J. Exp. Path., 32 (t95 I) 975 5 I . C. L A N N I N G AND S. S. C O H~EN, E N J. Biol. Chem.. I89 ( t 9 5 Q 1o9. {i F. L I P M A N N , Nature, i38 (i936) 588. 7 H. Me ILLWMN, J. Gen. Microb. 9b., 2 (1948) 288. AN, M. l". ITTER AND ( . tq. ~VERKMAN, lotva Slate Coll..]. £'ci. ~4 8 ~V. P . ~¢VIGGERT, M. SILVERMAN 0 9 4 o) I79. R e c e i v e d D e c e m b e r -'9tlI, ~953 i S. J. a F. D.