I R e p r i n t e d f r o m t h e J o u r n a l o f t h e A m e r i c a n C h e m i c a l S o c i e t y , 1 0 1 ,5 8 2 9 ( 1 9 7 9 ) . ]
Copvright r-c)1979 by the American Chemical Society and reprinted by peimission of the copyright owner
Conversionof the AdenosineMoieties
of RNA into ATP for Use in Cofactor Recycling
Sir.'
We reportherea practicalprocedurefor transformingthe
adenosinc
moieties
of RNA intoATP (SchemeI). YeastRNA
a n dt her equis it enz
e y meas rec o m m e rc i a l layv a i l a b l e
andi n(AcP) is easily
cxpensive,
and diammoniumacetylphosphate
p re par ed.Tr he m ix tu reo f n u c l e o ti d egse n e ra tebdy t hi sprocedure(containing
ATP, GMP, UMP, CMP, andotherminor
co ns t it uent sm) ay b e u s e dw i th o u tp u ri fi c a ti o ni n syntheti c
involvingenzymaticcatalysiswith ATP recycling.2.l
schemes
This methodis the mostpracticalonepresentlyavailablefor
g e n er at ing
t hc A T P rc q u i re di n s u c hs c h e me sT.h c fol l ow i ng
d e tailsillus t r at et he m a n i p u l a ti o ni sn v o l v e di n p re p ari ngthe
ATP - c ont aining
m ix tu re ,a n di n i ts u s ei n AT P -re q u i ri ng
enzymat ics y nt hes is .
In a rcprcscntative
procedure,
RNA (100g, 85%pure,from
, igm aC h e mi c aC
To r ulay eas tS
l o .) w a sd i s s o l v eidn 400 mL
o f w at er ,andt hes ol u ti o na d j u s te to
d p H 5 .6w i th N a OH and
t o 0 . 1m M i n Z n ( l l ) . 4N u c l e a sPer s( 8 . C .3 . 1 . 4 . - , 0 . m
2 0g , 9 3
w a sa l l o w e dt o s t i r a t 6 5 o C .
U 6 ) w a sa d d e da n dt h es o l u t i o n
S c h e m eI . C o n v c r s i o no t ' R N . A i n t o a M i r t r r r e o t ' N u c l c o t i d c s
C o n t a i n i n i rA T P
RNA
I N u c L ePa rs e
I
I
I
(Al1P+ Gl'lP+ U N P + C f ' 1 P
+...)
AcernrE
Krrunse
AoErvureKrruRse
K
originally containing,inter alia,l0glucose( I .0 mol), hexokinase
( E . C . 2 . 1 . 1 1 ,1 2 0 U ) , a n d a c e t a t ek i n a s e( 1 0 0 U ) i n 1 . 6 L o f
s o l u t i o n .A d d i t i o n o f A c P o v e r l 3 6 h , f o l l o w e d b y w o r k u p a s
describedpreviously,2yielded I I 0 g of solid,of which 90Vo(by
w e i g h t ) w a s a c c o u n t e df o r b y e n z y m a t i c a s s a ya s B a G - 6 - p .
7 H z O . T h e y i e l d o f G - 6 - P b a s e do n A c P w a s 5 l % . S i m i l a r l y ,
1 7 0 g ( 4 2 0 m m o l , 9 l 7 o p u r e b y w e i g h t , T } V a b a s e do n a c e t y l
phosphate)of dicyclohexylammoniumsr-glycerol 3-phosphate
w a s i s o l a t e dl o l l o w i n g p r o c e d u r e sd e s c r i b e de l s e w h e r e la f t e r
l 2 h o f o p e r a t i o no f a r e a c t o r o r i g i n a l l y c o n t a i n i n g g l y c e r o l
( - 5 0 0m m o l ) . g l y c e r o lk i n a s e( 8 0 0 U , F , . C . 2 . 7 . 1 . 3 0 )a, c e t a t e
k i n a s c( 4 2 0 L J ) ,a n d a 9 0 - r n L a l i q u o to i t h c n u c l e o t i d es o l u t i o n
( 1 . 7 m n r o l o f A T P ) . B o t h o f t h c s er e a c t i o n sf o l l o w e da c o u r s e
w h i c h w a s q u a l i t a t i v e l vi n d i s t i n g u i s h a b lfer o m t h a t o b s e r v c d
w i t h s i m i l a r r c a c t i o n sc a r r i e do u t w i t h p u r e A T P : t h i s o b s e r v a t i o n c s t a b l i s h e tsh a t t h e c r u d c , A T P - c o n t a i n i n g ,n u c l e o t i d e
s o l u t i o ns c r v e sa s a s a t i s f a c t o r ys u b s t i t u t ef o r p u r e A T P .
M u c h o f t h e e x p e n s eo f c o m m er c i a l A T P i s d e t e r m i n e db y
i t s p u r i f i c a t i o n . t l i g h p u r i t l i s n o t n e c e s s a r yf o r c o f a c t o r st o
b e u s e di n r c c r c l i n gs c h e n t e sb.u t a n v i m p u r i t i e sm u s t n o t i n terfere with thc rcactionsnor degradethe enzymes,cofactors,
r e a c t a n t s ,o r p r o d u c t s .T h e m i x t u r e o f n u c l e o t i d e sg e n e r a t e d
by this procedurecontains no more Lhan27 molVo ATP, but,
s i n c en o p u r i f i c a t i o ns t e p sa r e r e q u i r e dp r i o r t o i t s u s e i n p r o ceduresinvolving cofactor recycling.it providesa particularly
c o n v e n i c n ts o u r c eo f . \ T P f o r o r g a n i cs y n t h e t i cu s e .W c n o t e ,
h o w e v e r t, h a t p u r c , A T Pc a n b c i s o l a t e df r o m t h i s m i x t u r e b y
c o n v e n t i o n atlc c h n i q u e si.f r e q u i r e d .T h i s p r e p a r a t i o no f A T P
is superior to one reported earlier based on phosphorylation
o f a d e n o s i n e : 1t0h e e n z y m e s r e q u i r e d a r e a l l c o m m e r c i a l l y
a v a i l a b l e ,a n d t h c c o s t o f t h c s t a r t i n ga d e n o s i n em o i e t i e si s
lower.
Acknowledgments. This research was supported by the
N a t i o n a l I n s t i t u t e so i H e a l t h .G r a n t G M 2 6 5 4 3 .
A^D
AC
(ATP + Gl'lP+ U l , 1 P+ C i \ , l P+ . . . )
The progress
of the digestionwasfollowedby LC7 and by enzym at icas s ayf or AMP Ua n d a p p e a re d
to b e c o m p l etei n 65
h . T h e r e s u l t i n sg o l u t i o nc o n t a i n c (dm i l l i m o l e sA) M p ( 8 0 ) ,
G M P ( 6 0 ) ,U M P ( 8 l ) , a n dC M P ( 3 l ) . A c t i v ee n z y m ce o u l d
be recovered
from the digestby ultrafiltrationand reused.
A n aliquotof t hiss o l u ti o n(2 8 0mL , 5 0 mmo lo f A M P ) w as
co o ledt o r oomt emp e ra tu re
a n dd i l u te dto 2 .2 5L . T he pH of
th es olut ionwasadj u s te dto p H 7 .6w i th 2 M N a 2 C O 3,anda
p H- s t atwasem plo y e dto ma i n ta i nth i s p H th ro u g hthe subscq uentr eac t ionby a d d i ti o no f 2 M N a uC O 3 .A c e tateki nase
( E C . 2 . 1. 2 . 1 ,1 0 0U ) a n da d e n y l a tkei n a s c( 8 . C .2 . j . 4 . 3 , 3 0 0
U) immobilizedin -200-prn particlesof cross-linked
poly(a c r y lam ide- c o- N -a c ry l o x y s u c c i n i mgi e
d lee) w e re added,
togetherwith magnesium
acetate(75 mmol),ATP (0.I mmol),
a n ddit hiot hr eit ol
( 0 3 g ) D i a mmo n i u m
( 1.0
a c e rypl h o s phate
M solut ion)wasadd e da t 2 .2mmo lh -r to th es ti rrc ds ol uti on.
Ai ter 44 h of addit i o nth
, c re a c ti o nmi x tu rec o n ta i n ed
(mi l l i m o l c s )A T P ( 4 1 ) ,A D P ( 5 ) ,a n dA M P ( I ) . 8T h c s eq u a n t i t i e s
correspond
to a 907oselectivityfor conversion
of acetylphosp h at eint o A T P andA D P . T h e re m a i n d eor f th e a c etylphosp h at ewaslos tby hy d ro l y s i sn;o d i - o r tri p h o s p h a tes
of other
nucleotides
wercdetected.
This mixturewasuseddirectlyin enzyme-catalyzed
reacti o n sr equir ingA T P re c y c l i n gF. o r e x a mp l ea, n a l i quot(250
mL) wasusedto provide7 mmolof ATP for a reactionmixture
Referencesand \iotes
( 1 ) L e w i s ,J . M . ; H a y n i e S
, . L . ; W h i t e s i d e sG, . M . J . O r g .C h e m . 1 9 1 9 , 4 4 ,
864-865.
( 2 ) P o l l a kA
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2366-2367
( 3 ) R i o s - M e r c a d i l lVo . M : W h i t e s i d e sG
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(5) Kuninaka,A.; Otsuka,S.; Kobayashi,Y.; Sakaguchi,K. Butt.Agr. Chem.
Soc. Jpn. 1959,23, 239-243.Kuninaka,A.; Kibi,M; Yoshino,H.; Sakaguchi,K. Agr. Biol. Chem. 1961,25,693-701. Ogata,K. Adv. Appl. Microbiol.1975. 79.209 248
( 6 ) O s h i m aT, . ; U e n i s h iN
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7
8
3
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777
(7) LC analyzeswere carriedout usinga C-18 trr-BoTrdapac
reverse-phase
columnwith 5 mM tetrabutylammonium
phosphate,pH 7 .2 (WatersAssociates).
(8) Keppler,D. ln "Methodsof EnzymaticAnalysis",2nd Englished., Bergm e y e r ,H . U . , E d . ;A c a d e m i cP r e s s :N e w Y o r k , 1 9 7 4 ;V o l . 4 , p p 2 0 8 8 2096.
(9) Pollak,A.; Baughn,R. L.; Adalsteinsson,
O.;Whitesides,G. M. J. Am. Ctcm.
Soc. 1978, 100,303-304.Whitesides,
G. M.;Lamotte,A.; Adatsteinsson,
"Methods
O.;Colton,C. K. In
in Enzymology",
MosbachK., Ed.;Academic
P r e s sN
: e w Y o r k , 1 9 7 6 ;Y o l . 4 4 , p p 8 8 7 - 8 9 7 .
(10) Metal ion and antioxidantconcentrations
were thosedescribedin more
detailedprocedures.2,3
Baughn,R. L.; Adalsteinsson,
O.; Whitesides,G.
M. J. Am. Chem.Soc. 1978, 700,304-306.
( 11 ) N A T O / D A A D
p o s t d o c t o r af el l l o w ,1 9 7 8 - ' 1 9 7 9 .
(12) CONACyT(Mexico)predoctoralfellow.
Hans-JiirgenLeuchs,ll Jerome M. Lewis
Victor M. Rios-Mercadillo,l2GeorgeM. Whitesides*
Depart ment of Chemistry
' ""
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ReceiuedJune l. 1979