α-l-Formamidinoglutaric acid as a formylating agent
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278 SHORT C()MMIrNI(LVrlONS, t)RELIMINARY NOTES V()L. 17 (1055) work do not show any stimulation upon tile addition of :XG or related compounds. It appears then logical to assume by analogy that the bacterial enzyme might possess an active center with a structure similar to acetyl glutamate or related compounds. If this is tile case two possil)ilitics are open, either that carbamyl phosphate remains attached to the enzyme until reaction with ornithine or other acceptor, or that carbamyl phosphate is split off from the enzyme site lmfore further regwtion. :\s shown in Table 1l the second possibility seems to be true. Still to be inxestigated is whether or not (:omt)ound X is split to free carlmmyl ph~sl)hate before reacting with ornithine or other acceptor and xxhether this splitting is enzymic or non- euzvnlic 3. .\ further subject for in\estigati<m will be to de/ermine the possil)le existence of a "c;irhamyl or acety[ active Cellter" ill the "ll;Itive" lllHl/lluitli~ln ellzvine whi(h nl~ly be masked or denatlAred during the isolation ~)1 tile enzyme a. ~ANTIAGO (;RISOLIA * ~ 3lc llvai*l 14ivclwmical Cardiovascular l.atJoralories, l)@arhmud o/ :lledicil~e, HAROL1) J. (;RAI)V 17~liversily o/ I(a~sas 3h'dical ('c~Hcr, l(a*:sas ('ily, NaJtsas ((LS_.I.) I)()NALI) ]'.\VALI.A(?t[*** M. E. JoNEs, 1.. SPeCTeR AXn F. I.II,MAXN, J..tin. ('hem. Not., 77 (1055) 819. 2 ,% GRISOHA AN]) 1'. P. Com,:.~', .]. lli,I. (Tram., ~'o4 (1!)53) 753. a ,%. (;RISOHa aNn P. 1'. COHEN, .]. l~ioI. ('hem., 198 (1952) 5~I. 4 S. (;RISOLIA AND R. O. ~,IARNItAI.I., in \V. 1). Mcl':I.Rov AND B. (;LASS, Ami*w Acid 3lelab,dism, Johns Hopkins Press, Baltimore, 1955, p. 258. 5 S. (;RISOLIA, D. 1'. \~ALLACH AND 11. J. (;RADY, l¢iochim. Biot'ays. :-till, in press. 6 (). II. l.owRY AND J. ~\. l.Ol,l,~Z,./, l¢icd. (;hem., 102 (1940) 421. 7 (). E. Ih;-PLER, 3 l a m u d o/ Cli~Hcal /.al~orah>ry ,llettto<ls, Charles C. "l'h<mms, Springliehl, Illinois, 191c~ - P- "73. J. ('. 'I'O\VNI~ AND ,~. (;RISOLIA, unpublished. Received April 13th, 1955 • The following abbreviations are used throughout this paper : AG, acetyl glutainate ; CP, carbainyl phosphatel; Compound X, the active carbamyl phosphate internmdiate formed from :\(; or related compounds"; ATP, adenosinetrit)hosphate. 2\11 analytical methods and procedures have been de- scribed in preceding papersa, a. • *'l'his work was done during the tenure of an l'2staMished lnvestigatorshi t) of tile American t leart Association. • ** I'ost 1)octorute I:ellow of tile National lleart Institute, National Institutes of tlealth. a-L-Formamidinoglutaric acid as a formylatin 9 agent* The isolation of a4:fornmmidinoglutaric acid (I,':\G) 1,u,a,4 from incubation mixtures of histidine or tlrocallic acid with mammalian liver preparations suggested that FAG nlight be an intermediate in the pathway of the metabolisnl of histidine as a donor of the one carbon unit. Since in these enzymic digests FAG accumulated and no further degradation was observed, it was assumed that a suitable formyl acceptor was absent in these extracts 1, although the possibility could not be excluded that activation of histidine as formyl donor followed a pathway other than that through urocanic acid and FA(; s. \Ve wish to report two lines of evidence which strongly suggest that FAG or a compound in equilil/rimn with it is an obligatory intermediate in reactions in which histidine acts as a formylating agent. When I;A(; (5 pAJ) was incubated with_ folic acid (0.25 [13I) and 0. 5 Inl of a soluble enzyme extract from rat liver (supernatant of 1 : l liver homogenate with o.o 5 M phosphate buffer, pit 7.2, centrifuged at 14o,ooo >(, g at o 4 for 2 hours) in a total w/lume of o.8 ml, a sky-blue-fluorescing compound was formed which behaved identically with synthetic Io-formylfolic acid 6 upon paper chromatograt)hy with six solvents and ionophoresis. Under these conditions total conversion of lolic acid to "forinyl-folic acid" was obtained within an incul)atiou time of 3 hours at 37" in the darlc. The enzyme extract was inactivated by treatment with Dowex-24;l or prolonged dialysis aud could be reactivated to a varying degree by the addition of o.2 ml of boiled (2 rain, I oo') enzyme extract. The efficacy of tile boiled enzyme was abolished by treatment with l)owex-2-C1 or charcoal. The activity of tile inactivated enzyme could also be restored by folic acid which had been reduced either with Adams' catalyst ill OA N NaOI-U or with NaBH.t. The enzymic formation of "formylfolic * This work was supported in part by a grant from tile National Institute of Neurological Disease and l-~lindness ((;rant H-220) of the National Institutes of Health, l'ublic Health Service. Taken fn)m a doctoral dissertation to t)e sutmlitted by ALEXANDER MILLER.
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Transcript of α-l-Formamidinoglutaric acid as a formylating agent
278 SHORT C()MMIrNI(LVrlONS, t)RELIMINARY NOTES V()L. 17 (1055)
w o r k do n o t s h o w a n y s t i m u l a t i o n u p o n t i l e a d d i t i o n of :XG or r e l a t e d c o m p o u n d s . I t a p p e a r s t h e n log ica l to a s s u m e b y a n a l o g y t h a t t he b a c t e r i a l e n z y m e m i g h t possess an a c t i v e c e n t e r w i t h a s t r u c t u r e s i m i l a r to a c e t y l g l u t a m a t e or r e l a t e d c o m p o u n d s . If t h i s is t i le case t w o poss i l ) i l i t i c s a re open , e i t h e r t h a t c a r b a m y l p h o s p h a t e r e m a i n s a t t a c h e d to t h e e n z y m e u n t i l r e a c t i o n w i t h o r n i t h i n e or o t h e r a c c e p t o r , or t h a t c a r b a m y l p h o s p h a t e is sp l i t off f rom t h e e n z y m e s i t e lmfore f u r t h e r regwtion. : \ s s h o w n in T a b l e 1l t h e s econd p o s s i b i l i t y s e e m s to be t rue .
St i l l to be i n x e s t i g a t e d is w h e t h e r or n o t ( : omt )ound X is sp l i t to free c a r l m m y l p h ~ s l ) h a t e be fo re r e a c t i n g w i t h o r n i t h i n e or o t h e r a c c e p t o r a n d xxhether t h i s s p l i t t i n g is e n z y m i c or non- e u z v n l i c 3 .
.\ f u r t h e r s u b j e c t for i n \ e s t i g a t i < m wi l l be to d e / e r m i n e t he poss i l ) le e x i s t e n c e of a " c ; i r h a m y l or a c e t y [ a c t i v e Cel l ter" ill t h e " l l ; I t i v e " lllHl/lluitli~ln e l l zv ine w h i ( h nl~ly be m a s k e d or denat lAred d u r i n g t he i s o l a t i o n ~)1 t i l e e n z y m e a.
~ANTIAGO (;RISOLIA * ~ 3 l c llvai*l 14ivclwmical Cardiovascular l.atJoralories, l)@arhmud o/ :lledicil~e, HAROL1) J. (;RAI)V
17~liversily o/ I(a~sas 3h'dical ('c~Hcr, l(a*:sas ('ily, NaJtsas ((LS_.I .) I)()NALI) ]'.\VALI.A(?t[***
M. E. JoNEs , 1.. SPeCTeR AXn F. I.II,MAXN, J . . t i n . ('hem. Not., 77 (1055) 819. 2 ,% GRISOHA AN]) 1'. P. Com,:.~', .]. l l i ,I . (Tram., ~'o 4 (1!)53) 753. a ,%. ( ;RISOHa a N n P. 1'. COHEN, .]. l~ioI. ('hem., 198 (1952) 5~I . 4 S. (;RISOLIA AND R. O. ~,IARNItAI.I., in \V. 1). Mcl ' : I .Rov AND B. (;LASS, Ami*w Acid 3lelab,dism,
J o h n s H o p k i n s Press, B a l t i m o r e , 1955, p. 258. 5 S. (;RISOLIA, D. 1'. \~ALLACH AND 11. J. (;RADY, l¢iochim. Biot'ays. :-till, in press . 6 (). II . l . owRY AND J. ~\. l.Ol,l,~Z,./, l¢icd. (;hem., 102 (1940) 421. 7 (). E. Ih;-PLER, 3 lamud o/ Cli~Hcal /.al~orah>ry ,llettto<ls, Char l e s C. "l'h<mms, Sp r ing l i eh l , I l l ino is ,
1 9 1 c ~ - P - " 7 3 . J. ('. 'I'O\VNI~ AND ,~. (;RISOLIA, u n p u b l i s h e d .
R e c e i v e d A p r i l 13th, 1955
• T h e f o l l o w i n g a b b r e v i a t i o n s are u s e d t h r o u g h o u t t h i s p a p e r : AG, a c e t y l g l u t a i n a t e ; CP, c a r b a i n y l p h o s p h a t e l ; C o m p o u n d X, t h e a c t i v e c a r b a m y l p h o s p h a t e i n t e r n m d i a t e f o r m e d f rom : \ ( ; or r e l a t e d c o m p o u n d s " ; ATP, a d e n o s i n e t r i t ) h o s p h a t e . 2\11 a n a l y t i c a l m e t h o d s a n d p r o c e d u r e s h a v e b e e n de- s c r i b e d in p r e c e d i n g papersa , a.
• * ' l ' h i s w o r k was done d u r i n g the t e n u r e of an l '2s taMished l n v e s t i g a t o r s h i t) of t i l e A m e r i c a n t l e a r t A s s o c i a t i o n .
• ** I 'os t 1 )oc to ru te I :e l low of t i le N a t i o n a l l l e a r t I n s t i t u t e , N a t i o n a l I n s t i t u t e s of t l e a l t h .
a-L-Formamidinoglutaric acid as a formylatin 9 agent*
The i s o l a t i o n of a 4 : f o r n m m i d i n o g l u t a r i c ac id (I, ':\G) 1,u,a,4 f rom i n c u b a t i o n m i x t u r e s of h i s t i d i n e or t l roca l l i c a c i d w i t h m a m m a l i a n l i ve r p r e p a r a t i o n s s u g g e s t e d t h a t FAG n l i g h t be an i n t e r m e d i a t e in t h e p a t h w a y of t h e m e t a b o l i s n l of h i s t i d i n e as a d o n o r of t h e one c a r b o n u n i t . S ince in t he se e n z y m i c d i g e s t s F A G a c c u m u l a t e d and no f u r t h e r d e g r a d a t i o n w a s o b s e r v e d , i t w a s a s s u m e d t h a t a s u i t a b l e f o r m y l a c c e p t o r w a s a b s e n t in t h e s e e x t r a c t s 1, a l t h o u g h t h e p o s s i b i l i t y cou ld n o t be e x c l u d e d t h a t a c t i v a t i o n of h i s t i d i n e as f o r m y l d o n o r fo l l owed a p a t h w a y o t h e r t h a n t h a t t h r o u g h u r o c a n i c ac id a n d F A ( ; s.
\Ve wish to r e p o r t t w o l ines of e v i d e n c e w h i c h s t r o n g l y s u g g e s t t h a t F A G or a c o m p o u n d in e q u i l i l / r i m n w i t h i t is a n o b l i g a t o r y i n t e r m e d i a t e in r e a c t i o n s in w h i c h h i s t i d i n e a c t s as a f o r m y l a t i n g a g e n t . W h e n I ; A ( ; (5 pAJ) w a s i n c u b a t e d with_ fol ic ac id (0.25 [13I) a n d 0. 5 Inl of a so lub l e e n z y m e e x t r a c t f rom r a t l i v e r ( s u p e r n a t a n t of 1 : l l i v e r h o m o g e n a t e w i t h o.o 5 M p h o s p h a t e buffer , p i t 7.2, c e n t r i f u g e d a t 14o,ooo >(, g a t o 4 for 2 hours ) in a t o t a l w / l u m e of o.8 ml , a s k y - b l u e - f l u o r e s c i n g c o m p o u n d w a s f o r m e d w h i c h b e h a v e d i d e n t i c a l l y w i t h s y n t h e t i c I o - f o r m y l f o l i c ac id 6 u p o n p a p e r c h r o m a t o g r a t ) h y w i t h s ix s o l v e n t s and i onophore s i s . U n d e r t h e s e c o n d i t i o n s t o t a l c o n v e r s i o n of lo l ic ac id to " f o r i n y l - f o l i c a c i d " was o b t a i n e d w i t h i n an i n c u l ) a t i o u t i m e of 3 h o u r s a t 37" in t h e darlc. T h e e n z y m e e x t r a c t w a s i n a c t i v a t e d b y t r e a t m e n t w i t h D o w e x - 2 4 ; l or p r o l o n g e d d i a l y s i s a u d cou ld be r e a c t i v a t e d to a v a r y i n g d e g r e e by t h e a d d i t i o n of o.2 ml of bo i led (2 rain, I o o ' ) e n z y m e e x t r a c t . The ef f icacy of t i l e bo i l ed e n z y m e was a b o l i s h e d b y t r e a t m e n t w i t h l )owex-2-C1 or c h a r c o a l . T h e a c t i v i t y of t i l e i n a c t i v a t e d e n z y m e cou ld a lso be r e s t o r e d b y folic ac id w h i c h h a d been r e d u c e d e i t h e r w i t h A d a m s ' c a t a l y s t i l l OA N NaOI-U or w i t h NaBH. t . The e n z y m i c f o r m a t i o n of " f o r m y l f o l i c
* T h i s w o r k w a s s u p p o r t e d in p a r t b y a g r a n t f rom t i l e N a t i o n a l I n s t i t u t e of N e u r o l o g i c a l D i s e a s e a n d l-~lindness ( ( ; r a n t H-220) of t h e N a t i o n a l I n s t i t u t e s of H e a l t h , l ' ub l i c H e a l t h Service . T a k e n f n ) m a d o c t o r a l d i s s e r t a t i o n to t)e s u t m l i t t e d b y ALEXANDER MILLER.
W)L. 1 7 ( 1 0 5 5 ) SHORT COMMUNICATIONS, PRELIMINARY NOTES 2 7 9
a c i d " w a s n o t i n h i b i t e d b y v e r s e n e or b y ae rob i c c o n d i t i o n s a n d d id n o t r e q u i r e a d e n o s i n e t r i p h o s - p h a t e . F o r m i c ac id , formyl - i soglu tamine 1, - g l u t a m i n O a n d - g l u t a m i c ac id l were i n a c t i v e , w h i l e u r o c a n i c ac id w a s as a c t i v e as F A G o n l y a t h i g h c o n c e n t r a t i o n s (5 1~3I) a n d h i s t i d i n e w a s s t i l l less a c t i v e * . T h e s u p e r i o r i t y of F A G to u r o c a n i c ac id or h i s t i d i n e as t ' o rmyl d o n o r was c l e a r l y d e m o n - s t r a t e d w h e n fo rmic a c i d - n C (25 ii.,l'l) w a s i n c u b a t e d w i t h p i g e o n l i ve r e x t r a c t s n n d e r t i le c o n d i t i o n s u s e d b y {~()LIYFII\VAIT el al. s to r t i l e e n z y m i c s y n t h e s i s of f o r m y l g l y c i n a m i d e r i b o t i d e . Af t e r r i g o r o u s r e m o v a l of al l v o l a t i l e r a d i o a c t i v e m a t e r i a l 14,'; c< ,unts /min , c o r r e s p o n d i n g to a u t i l i z a t i o n of o. 5 lo l l of fo rn la te , were found in t i le n o n v o l a t i l e f r a c t i o n in t i l e p r e s e n c e of formic ac id a lone, lTl/on a d d i t i o n of I o t ~ l l e a c h of non la l ) e l ed h i s t i d i n e , u r o c a n i c ac id , FAG, f o r n l y l i s o g l u t a n l i n e , f ( w m y l g h l t a m i n e , i ' o r m y l g l u t a n l i c m i d , f (~rmylglycine, f o r m a m i d i n o a c e t i c ac id :l, a n d ~ - l l enzy l F . \ ( ; a, 12o, St). 48, I35 , I -'~>, 137, 155 a n d t_'5 cCmnts /min , r e s p e c t i v e l y , were found in t he n o n v o l a t i l e f r ac t ion . The i n c r e a s i n g i n h i b i t i o n of i n c o r p o r a t i o n of fo rmic a c i d i n t o t h e n o n v o l a t i l e f r a c t i o n as one p r o c e e d s f rom h i s t i d i l w to FAG as well as t he l ack of i n h i b i t i o n b y o t h e r f o r m y l d e r i v a t i v e s g i v e s s t r o n g supp(wt to lilt" ro le of FAG as sou rce of t h e f o r m y l g roup . The e x t e n t of i n h i b i t i o n s h o w s t h a t 1;:\(; does n o t a c t as a f o r l n y l a t i n g a g e n t t h r o u g h t h e p r o d u c t i o n of f o r m a t e . T h e s a m e c l m c l u s i o n w a s r e a c h e d w h e n l abe l ed h is t id ine-- ' -zaC ** (e. 5 / ~ M ) w a s i n c u b a t e d u n d e r t h e a b o v e c o n d i t i o n s in t he a b s e n c e or p r e s e n c e of a 4-fold excess of n o n l a b e l e d f o r m a t e . A f t e r d e p r o t e i n i z a t i o n , h i s t i d i n e w a s r e m o v e d on al l ) o w e x - 2 - f o r n m t e c o l u m n a n d t h e e l u a t c b r o u g h t to d r y n e s s a n d c o n n t e d . [n t he p r e s e n c e ~)f f o r m a t e an i n h i b i t i o n of i n c o r p o r a t i o n of u(- of o n l y 4o01, was o b s e r v e d in t h e n o n v o l a t i l e , 1t('1 l ab i l e s, f r ac t ion .
The m e c h a n i s m of t h e m e t a b o l i c u t i l i z a t i o n of I~'AG as f o r m y l d()nor as wel l as t h e de t a ih !d r e q u i r e l n e n t s (if t i le s()lul/le enzyBle s y s t e m fire n l l de r s t u d y .
:\LEXANIIER ~I ILLER Department el l¢ioehemistry, College o~ Phvsicia*zs a~d Surgeons, ] tEINRICH \VAEI.SCH
( 'ohtmbia Universily, Nea.~ Yor/e, a*td the N e w ~'()rl; Stale Psychiatric hzstil*th', New York, N . ~ . ( l : .N .q.)
1 l]. : \ . I{OREK AND l I . \V.,xELSCH, J. Biol. Chem., 205 (1953) 459. '-' 11. TABOR AND A. If . ,~IEttLER, J. l~iol. Chem., 2 t o (L954) 559- a A..~IILLER AND H. \VAELSCH, .]..4~1. Chem. Soc., 76 (1954) 0195. 4 j . E. S~,:EGmLLER, M. SILVERMAN, [ t . TABOR AND A. H. MEHLER, J . A m . Chem. See., 76 ([954) 6205.
If. \VAt,.'LSCH AND A. MILLER, .q miml Ac id Metabolism, \V. D. McELROY AND \V. 1~. GLASS (Fd i to r s ) , J o h n s t t o p k i n s l ' ress , B a l t i m o r e , M a r y h m d , 1955, p. 4o7 •
t~ M o d i f i c a t i o n of t h e p r o c e d u r e of M. (;ORDON, J. M. RAVEL, R. E . EAKIN AND \V. S tovE , .]. A m . Chem. Nee., 7 ° ( I948) 878.
v 1',. L. O'I)ELL, J. ~'l. VANllENBELT, E. S. I~LOOM AND J. J. PFIFFNER, .]r..'tltl. Cllet~l. SOC., 69 (1947) 25o- s I). :\. (;OLIYrtlWAIT, R. A. I'EABOI/V AND (;. l)t. (;REENI~ERG, J . . l l t~ . C/letll. Sou., 70 (~954) 5258.
R e c e i v e d A p r i l 1st, I955
• \ \ : h i l e t h i s w o r k w a s in p r o g r e s s K. SLAWK aND V. MATOULKOVA {Colleclicm ('zechoslov. Chem. (:,unmltn., I0 (1954) 1o32) r e p o r t e d t he f o r m a t i o n of f o r m v l f o l i c ac id , i d e n t i f i e d b y p a p e r c h r o m a t o - g r a p h y , u p o n i n c u b a t i o n for 22 h o u r s of c o n c e n t r a t e d l i v e r h o m o g e n a t e s w i t h folio ac id a n d h i s t i d i n e or u r o c a n i c ac id or an i o n o p h o r e t i c a l l y s e p a r a t e d e n z y m i c d e g r a d a t ; o n p r o d u c t of h i s t i d i n e a s s u m e d to be [ ; \ G . In s t i m u l a t i n g t o r m y l f o l i c ac id f o r m a t i o n t i le F.XG p r e p a r a t i o n p r o v e d to be s u p e r i o r to h i s t i d i n e or u r o c a n i c a c i d a n d o t h e r fo r lny l c~ ,mpounds w e r e f o u n d to be i n a c t i v e .
• * \Ve wish to t h a n k l)r. I). SPRINSON for t i le sn inp l e of L-hist idine-- ' -14C u s e d in t h i s work .
On the nature of the carbamyl group donor in citrulline biosynthesis*
O t / t i m m n c o n d i t i o n s for t i l e b i o s y n t h e s i s of c i t r u l l i n e f rom o r n i t h i n e , a m m o n i a , a n d c a r b o n d i o x i d e b y so lub le m a n m m l i a n l i v e r e n z y m e s r e q u i r e A T P * *, n l ag l l e s iu l l l lo l l s a n d c a t a l y t i c a n l o u n t s of c e r t a i n d e r i v a t i v e s of g l u t a m i c ac id 1. T h e c a t a l y t i c p r o p e r t i e s of t h e g l u t a m a t e d e r i v a t i v e s h a v e b e e n a s c r i b e d to t h e f o r m a t i o n of an u n s t a b l e c a r b a m y l i n t e r m e d i a t e ( C o m p o u n d X) c o n t a i n i n g t he g l u t a n m t e d e r i v a t i v e , a m m ( m i a , c a r b o n d i o x i d e a n d p h o s p h a t e in a m o l e - t o - m o l e r a t i o 1,2,a. T r a n s f e r of t he c a r b a m y l g r o u p from C o n l p o u n d X to o r n i t h i n e to fo rm c i t r u l l i n e was c o n s i d e r e d to r e s u l t in t i le r e g e n e r a t i o n of t l le g l u t a m a t e d e r i v a t i v e a n d t h e r e l ea se of i n o r g a n i c p h o s p h a t e .
* T h i s i n v e s t i g a t i o n w a s s u p p o r t e d i n p a r t b y a r e s e a r c h g r a n t f rom t h e N a t i o n a l I n s t i t u t e of A r t h r i t i s a n d M e t a b o l i c D i s e a s e s of t h e N a t i o n a l I n s t i t u t e s of H e a l t h , P u b l i c H e a l t h Serv ice , a n d t h e \V i scons in A l u n l n i R e s e a r c h l ; o u n d a t i o n .
* * T h e f o l l o w i n g a b b r e v i a t i o i i s a re u s e d : ATI ' , a d e n o s i n e t r i p h o s p h a t e ; AG, a c e t y l g l u t a m a t e ; C(;, c a r b a m y l g l u t a m a t e ; A G [ a n d CGI, i n t e r m e d i a t e s f o r m e d in t i l e p r e s e n c e of AG a n d (i(_; re- s p e c t i v e l y .
