[Reprinted from the Journal of the American Chemical Society, 100, 304 (19?8).]
Copyright 1978 by the American Chemical Society and reprinted by permission of the copyright owner
Large-ScaleEnzyme-Ciatalyzed
Synthesis
of ATP from Adenosineand Acetyl Phosphate.
Regeneration
of ATP from AMPI
Sir.'
In p re v i o u sre ports,w e have descri bedl arge-scal ene
z,yme-catalyz,ed
organicsyntheses
requiringthe enzymatic
regeneration
of ATP from ADP andacetylphosphate.z,r
Many
i mp o rta n tb i o s y n theti reacti
c
onstransformA TP to A MP
ra th e rth a n AD P; a few generate
adenosi ne.a
H erew e summarizethe operationof a three-en
whichconzymesequence
v e rtsa d e n o s i nto
e ATP (S cheme
I): In thi sscheme.
A MP and
ADP areinvolved
bothasintermediates
in the phosphorylation
o f a d e n o s i nto
e ATP , and as partsof the catal yti ccofactor
u ti l i z a ti o nc y c l ew hi chconsumes
and regenerates
A TP . Thi s
S c h e m eI . C o n v e r s i o no f A d e n o s i n ct o A T P
Adenosine
Adenosine Kinose
ATP
AcP
3 enzymes
N e t : A d e n o s i r ' e+ 3 A c P
0002-7863178I 1500-0304$0
1.00/0
ATP + 3Ac
(gr1978 American Chemical Societv
305
Communications
to the Editor
a q u c o u sp h a s ct o 7 . 4 w i t h g l a c i a la c c t i ca c i d .T h e p h i r s e sw e r e
s e p a r a t c da, n d A T P w a s r e e x t r a c t e di n t o a n a q u e o u ss o l u t i o n
r v h o s ep H w a s a d j u s t e dt o l l . 5 w i t h N a O H . T h i s a q u e o u s
solutionwas neutralizcdwith acctic acid. and the dibarrutnsalt
o f A T P w a s p r e c i p i t a t c db y a d d i t i o no f s l i g h t l y) 2 c q u i v ( 7 7
g , 3 0 0 m m o l ) o f b a r i u m a c e t a t e .l s o l a t i o n r 6o f t h c p r e c i p i t r t t c
y i e l d e d 6 0 . 9 g o f w h i t c p o w d e r w h o s eA T P c o n t e n t b y e n z ) ' rnatic assaylTcorrcspondedto 77",4,Ba1ATP'4tlrO(-54nlrnol).
E
T h i s v a l u e r c p r e s e n t sa n i s o l a t e dy i c l d l o r A T P o i 3 6 % ,b a s e d
1a
, s e do n A c P . T h c a c t i v i t i c so l ' r e c o v o n a d e n o s i n ea n d l - 5 o b
qt
e r e d e n z y m e c o r r e s p o n dt o 9 3 o / o f t h e a d e n o s i n ek i n a s ea n d
I
o
J5o/oolthe irnmobilizedacctatekinascaddcd.Adenylatekinasc
(l,
()
r e c o v e r yw a s a l s o g o o d b u t w a s n o t d e t e r m i n e da c c u r a t e l y .
a
zso
T h i s s y n t h e s i cs s t a b l i s h ctsh r e c p o i n t sp e r t i n c n tt o t h c u s c
o f c n z l ' m a t i cc a t a l y ' s iisn o r g a n i cs v n l h c s i sF' i r s t 't h c c n z i r n l a t i c
r c g e n c r a t i o nt t l - A T P I ' r t l n t A M P p r o c c e d ss n l o o t h l v u s i n g
a c c t l l p h o s p h a t ca s t h c u l t i n l l t t cp h o s p h a t cd o n o r : c n z y n l c c a t a l v z c dr c a c t i o n sl l h i c h c o n v c r tc t l l ' a c t o r' \ T P t o A M P c a n
t h u s b c u s e dl ' o rp r a c t i c a l - s c a l cs 1 ' n t h c s c sS.c c o n d 'n l a n i p u l a t i o n o f a c o u p l c ds y s t c n ro l ' t h r e ec n z y m c sa s p l l r t o f a s r n t h c t i c
p r o c c d u r c p r c s c n t s n o s p c c i a l p r o b l e m s .T h i r d , e n z \ m l r t i c
2 40
80
160
p r o c c d u r c s s h o u l d n o u b c c o n s i d c r e da s a n a l t c r n a t i v c t t l
conventional chcrnicltl or fcrnlcntation methods for the
Time (h)
a n d o t h c r n u c l e o t i d e fsr o n l n u prcsent
duringconversron l a r g c - s c a l cs r n t h e s i so l ' A T P
(millimoles)
ol'nuclcosides
Figurel. Quantitics
c
l
c
o
s
i
d
c
s
.
l
s
o f a d e n o s i nt ocA T P .
E roo
Referencesand \otes
s e q u e n c ee s t a b l i s h e tsh e p o t e n t i a lo f o r g a n i c s y n t h e s e sb a s e d
on ATP rcgenerationfrom AMP and adenosine.and illustratcs
a r o u t e f o r t h c p r c p a r a t i o no f n u c l e o t i d e sf r o m n u c l c o s i d e s .
A t y p i c a l r e a c t i o nw a s r u n a t r o o m t e m p e r a t u r ei n a , 5 - [ f l a s k e q u i p p c dw i t h a p F { e l e c t r o d ea n d m a g n e t i cs t i r r i n g b a r .
doubly distilledwater was addcd ATP.
To I L of deoxygenated.
A D P , a n d A M P ( 5 0 m g c a c ha s t h c s o d i u ms a l t s -. 0 . 1 m m o l ) .
a d e n o s i n e( 4 0 g , 1 5 0 m m o l , n o t c o m p l e t el y s o l u b l e ) ,m a g n c s i u m a c e t a t e( 4 g . l 9 m m o l ) , a n d d i t h i o t h r e i t o (l D T T . 3 0 0 m g ,
1 . 9m m o l ) . A d e n o s i n ek i n a s c( E . C . 2 . 1 . 1 . 2 0 , 4 0U ) , a d e n y l a t e
k i n a s e( 8 . C . 2 . 1 . 4 . 3-, 5 5 0u ) , a n d a c e t a t ek i n a s c( 8 . C . 2 . 1 . 2 . 1 .
1 8 0 U ) , i m m o b i l i z e di n c r o s s - l i n k e p
d o l y a c r y l a m i d eg e l p a r ')
ticlcsw
. c r c a d d c da s u s u s p e n s i t il n . 5 5 0m l - o l ' w u t c r . i A n
pH
was
was
flask,
maintainedin the
and the
argon atmosphere
k c p t b c t w e c n6 . 7 a n d 6 . 9 b y t h e p H s t a t c o n t r o l l c da d d i t i o no l '
2 M s o d i u n rc a r b o n a t cD
. i a n r m o n i u ma c e t y lp h o s p h a t c l (00 . 5
M ) w a s p u m p e di n c o n t i n u o u s l ya t a r a t c o i 0 . 1 m o l p c r d a y .
A l t e r t h e f i r s t f e w h o u r s o f o p e r a t i o n ,c o n c e n t r a t i o n so f s u b s t r a t c se x c e c d e dt h e M i c h a e l i sc o n s t a n t so f t h e e n z y m e s . l l
A d d i t i o n a l D T T I 2 a n d m a g n e s i u m a c e t a t e l sw e r e a d d e d
d u r i n g t h e c o u r s eo f t h e r e a c t i o n .A n a d d i t i o n a l 5 0 0 U o f i m m o b i l i z e da c e t a t ck i n a s ew a s a d d e d a f t e r 1 2 0 h o f o p e r a t i o n ,
becausethe ratc of formation of ATP was,at this point. limited
b y t h c a c t i v i t yo f t h i s e n z y m e .T h e q u a n t i t i e so f A M P , A D P .
a n d A T P i n s o l u t i o n d u r i n g t h e c o u r s eo f t h e r e a c t i o n a r e
s u m m a r i z e di n F i g u r e l ; A f t e r 2 3 9 h . 1 2 5 m m o l o f A T P , 2 0
m m o l o f A D P , a n d 3 m m o l o i A M P w e r c p r e s e n t ; r tah c f i n a l
concentrationof ATP was 30 mM. Thc yicld of phosphorylated
a d e n o s i n ed e r i v a t i v e sw a s 9 8 0 1b, a s e do n a d e n o s i n oa n d 3 8 o 1 ,
b a s e do n a c e t y l p h o s p h a t ea d d e d .T h e e q u i l i b r i u m c o n s t a n t s
f o r t h e r e a c t i o n s i n S c h e m e I a r e s u c h t h a t c o n v e r s i o no f
adenosineto ATP should have beencomplete,and conversions
. eh e a d c n o s i n e
i n s r n a lfe r s c a l er e i r c t i o n sw e r ea s h i g h a s 9 4 o / u T
kinase used in the procedure described was, however, cont a m i n a t e d w i t h A T P a s e a c t i v i t y , a n d t h e o b s e r v e df i n a l c o n centrationsrepresenta competition betweenthc rates oi ATP
lormation and ATP hydrolysis.
T h c e n z y m e - c o n t a i n i n gg e l a n d a w h i t c p r e c i p i t a t e ( p r i m a r i l y m a g n e s i u mp h o s p h a t e )w e r e s e p r i r a t e df r o m t h c s o l u t i o n b y c e n t r i f u g a t i o n .A T P w a s e x t r a c t e d i n t o a n o r g a n i c
p h a s cc o n s i s t i n go f 5 7 ow : w o c t a d e c y l a m i n ci n I - p e n t a n o l l sb v
m i x i n g t h e p h a s e st h o r o u g h l y a n d a d j u s t i n g t h e p H o i t h e
( 1 ) S u p p o r t e db y t h e N a t i o n a l S c i e n c e F o u n d a t i o n ,G r a n t G l 3 4 2 8 4 O A a c knowledges parlial f inancial supporl from the lcelandic Science Foundation.
R.L.B.wis a postdoctoraltrainee under NIH TrainingGrant 5 T32 CA 09112
ul.
(2) A. Pollak, R. L Baugh, and G M. Whitesides, J. Am. Chem. Soc., 99' 2366
( 1 9 77 ) .
( 3 ) Y . - S S h i h a n d G M W h i t e s i d e s ,J . O r g . C h e m . , 4 2 , 4 1 6 5 ( 1 9 7 7 \
(4) "The Enzymes", Vol B.3rd ed, P D. Boyer, Ed.,Academic Press,New
Y o r k . N Y . , 1 9 7 3 : E . R . S t a d t m a n .C h a p t e r 1 : S . H . M u d d , C h a p t e r 4 . G . L '
C a n t o n i . A n n .R e v . B i o c h e m . , 4 4 , 4 3 5 ( 1 9 7 5 ) .
( 5 ) A d e n o s i n e k i n a s e w a s p a r t i a l l y p u r i fi e d f r o m b r e w e r ' s y e a s t ( s i g m a ) a c cording to a literature scheme.6 The yeast extract was subjected to ammonium sulfate fractionation, DEAE-sephadex chromatography, and
Sephadex G-75-120 chromatography to yield a preparation of specific
activity0.1 U/mg (1 U = 1 gmol/min'mg). Acetate kinase and adenylate
kinase (both from Sigma) had specif ic activities ot 220 and 2250 U/mg,
r e s p e c t i v e l y ,f o l l o w i n g a c t i v a t i o n w i t h D T T
(6) T. K. Leibach, G. l speiss, T. J. Neudecker, G. Peschke, G. Puchwein, and
G . R . H a r t m a n n , Z P h y s i o l .C h e m , 3 5 2 , 2 2 8 ( 1 9 7 1 ) .
(7) Enzymes were immobilizedusing the proceduredescribed in A. Pollak,
R. L. Baughn, O. Adalsteinsson,and G. M. Whitesides' J. Am Chem. Soc',
p r e c e d i n g p a p e r i n t h i s i s s u e . T h e i m m o b i l i z a t i o ny i e l d s a n d g e l v o l u m e s
employed were as follows: adenosinekinase (25%' 300 mL), adenylate
kinase (40% , 25 mL), and acetate kinase (40% ' 25 mL).
(8) The enzymes must be mixed, rather than used separately as catalysts in
Seouential reactions. AdenOsine kinase and acetate kinase must be present
simultaneously to catalyze the phosphorylation of adenosine with AcP as
the ultimate phosphate donor in a reaction regenerating ATP. The conversion of ADP to ATP by adenosine kinase is driven by the high equilibrium
constant for the acetate kinase catalyzed phosphorylation of AMP'e
(9) R. Cuputto in "The Enzymes", Vol 6,2nd ed, P. D. Boyer' H' Lardy, K'
Myrback, Ed., Academic Press, New York' N.Y., 1962, p 133; R S Langer,
C . R . G a r d n e r .B . K . H a m i l t o n ,a n d C . K . C o l t o n ,A I C \ E J , 2 3 ' 1 ( 1 9 7 7 )
(10) G. M. Whitesides,M. Siegel, and P. Garrett,J. Org. Chem ,40' 2516' (1975)'
The material used was 82-89% pure, with ammonium acetate, ammonium
phosphate, and acetamide as the principal impurities. The AcP solution
oC before addition to minimize hydrolysis
was keot at 4
.1
(1 1) For acetate kinase, Ka"p= 0.34 mM and Kae" = ' 1 mM (C A' Janson and
(
1
9
7
4
) ) .F o r a d e n y l a t ek i n a s e ,
2
4
9
,
2
5
6
7
B
i
o
l
.
C
h
e
m
.
,
W . W . C l e l a n d ,J .
Knop = 1.58 mM, Kaup = 0.5 mM, and Kalp = 0 3 mM (P' DeWeer and A'
G. Lowe. J. Biol. Chem., 248,2829 (1973); J. M. Blair, Eur' J. Biochem',
= 0 . 0 0 . 1m M ( T ' E . B a r m a n ,
1 3 , 3 8 4 ( 1 9 7 0 ) ) .F o r a d e n o s i n e k i n a s e , K a
.1969'
p
" E n z y m e H a n d b o o k ' ' , V o l . I , S p r i n g e r - V e r l a g ,N e w Y o r k , N Y ,
394).
(12) Adenosine kinase is inhibited by DTT. The DTT concentration was initially
maintainedat 1-2 mM to minimize this inhibition.when all of the adenosine
had been phosphorylated, the DTT concentration was increased to 5-10
mM. The total quantity of DTT used was 5.5 g (36 mmol).
(13) Magnesium acetate was added as necessary to maintain a level 20-40
mmol higher than the assayed amount of ATP to ensure that free magnesiumli) would be present for complexation with ADP and AMP. The total
quantity of magnesium acetate added was 36 g (168 mmol)' .
( 14) ioncentrations were assayed by standardenzymatic methods: Hans Ulrich
Bergmeyer, Ed., "Methods of Enzymatic Analysis", Verlag Chemie,
Welnneimlgergstr., Germany, Academic Press' New York and London,
1974.
(15) G. W. E. Plaut, S. A. Kuby. and H. A. Lardy, J Biol. Chem.' 243,184 (1950)'
The basic orocedure described in this reference for the extraction of ATP
3 06
J ournalrf the AmericanChemicalSociety / 100:1 f J anuary4, 1978
should result in a distributioncoeff icient of 1 1. lt was, however. found that
the presence of high concentrations of other salts in the reaction mixture
interfered with ATP extraction. As a result, the reaction mixture was diluted
oC (to
with 50% of its volume of water and then extracted twice at 45
maintain homogeneity) with volumes of organic phase equal to the original
volume. This treatment resulted in the separation of 75 % of the ATP from
the aqueous phase; 44'k ot this ATP was subsequently isolated.
(16) G. A. LePage in "Biochemical Preparations",Vol. l, H. E. Carter, Ed., Wiley,
N e w Y o r k , N Y . , 1 9 4 9 ,p 5 .
( 1 7 ) T h e p r e p a r a t i o na l s o c o n t a i n e d 7 % A D P ,b u t n o d e t e c t a b l e A M P . T h e r e mainder was presumably barium phosphate.
(18) The specificity of adenosinekinases is broad; cf. ref 6 and B. Lindberg,
H. Klenow, and K. Hansen, J Biol. Chem., 242,350 (1967). A large number
o f o t h e r n u c l e o s i d e sa n d n u c l e o t i d ek i n a s e s a r e a l s o k n o w n ; c f . E . P . A n derson in "The Enzymes", Vol. 9, 3rd ed, P. D. Boyer, Ed., Academic Press,
N e w Y o r k , N . Y . , 1 9 7 3 , C h a p t e r 2 . C e l l - t r e e e n z y m a t i c s y n t h e s i so f A T P
might be especially valuable when alternative synthetic routes yield product
mixtures containingdifficulty removed impurities(for example, GTP in
fermentation processes).
Richard L. Baughn,Orn Adalsteinsson
GeorgeM. Whitesidesx
Dep art ntent td Ch enti.st r "v'
lrl o.s.s
uch u.\et t .sI n.\t i t u t e o.f''f echnctI ogy
('amhridgt',M a.s.sut'hu.tett.t
02 I 39
Rct'eiretlSeptentber29, 1977