Reprinted from the Journal of the American Chemical Society, 1989, lt tJ 8920.
Chemical Soeiety and reprinted by permission of the copyright
O G85 bt tft"-A-""ican
Copyright
Preparation of Phosphoenolpyruvate from
D-(-)-3-Phosphoglyceric Acid for Use in Regeneration
Of ATPI
and
E t h a n S . S i m o n . 2S v e nG r a b o w s k i , 3
G e o r g eM . W h i t e s i d e s *
Departmentof Chemistrv, H art'ard Linit'ersit)'
02 138
Cambridge,M as,sachu.\ett.s
R e c e i r e dJ u l y 2 4 , 1 9 8 9
Adenosine5'-triphosphate(ATP) is the most usefulphosphoryl
d o n o r i n e n z y m e - c a t a l y z esdy n t h e s i s . 4 T h e m o s t c o n v e n i e n t
procedure for in situ regenerationof ATP from ADP uses
p h o s p h o e n o l p y r u v a(tP
e E P ) a n d p y r u v a t ek i n a s e( S c h e m eI ) .
e n d m u s t u s u a l l yb e s y n t h e s i z e d
C o m m e r c i a lP E P i s e x p e n s i v a
chemicallyfor use in large-scalesynthesis.Here we describea
practicalprocedure(the PGA method) that usestwo inexpensive,
commerciallyavailableenzymesto generatePEP in situ from the
relatively inexpensiveo-(-)-3-phosphoglycericacid (3-PGA)
( S c h e m eI ) . 6
of ATP UsingPEP,
SchemeI. Regeneration
x .o. 1H
PO\-//\/.O-
a-roo3
phosphoryfated
procluct
phosohoalvcerctel
-'
muta'se
,f
E.c27.s.3
I
ATP
|
o
tt
)\v.oll
pyr o
\
\
,
,/
/
oP
|
)---oeyruvatekinase
ll
E-.c.2.7.1.4o
K_10"
ll
pep o
Enolase
E'c'42111
^-'
-_
x '-l
.OP
"o-nf
,-rco
o
' P E P c a n b e s y n t h e s i z e di n a s e p a r a t ec h e m i c a ls t e po r g e n e r a t e da s
s h o w n f r o m 3 - P G A . P - p h o s p h a t e l3 - P G A = D - ( - ) - 3 - p h o s p h o g l y c e r i c a c i d ; 2 - P G A = D - ( + ) - 2 - p h o s p h o g l y c e r iacc i d ; p p p = p h o s p h o e n o l p y r u v a t ep; y r = p y r u v a t e .
( l ) S u p p o r t e db y N I H G r a n t G M 3 0 3 6 7 .
( 2 ) D u P o n t F e l l o w1 9 8 6 - 1 9 8 7 .
(3) NATO Postdoctoral
by the Deutscher
Fellow1988-1989(administered
AkademischerAustauschdienst).
( 4 ) C h e n a u l t ,H . K . ; S i m o n ,E . S . ; W h i t e s i d e sG, . M . I n B i o t e c h n o l o g y
& GeneticEngineeringReuiews:Russell,G. E., Ed.; Intercept: Wimborne,
D o r s e t ,1 9 8 8 ;V o l . 6 , C h a p t e r6 .
( 5 ) H i r s c h b e i nB, . L . ; M a z e n o d F
, . P . ; W h i t e s i d e sG, . M . J . O r g . C h e m .
1982.47.3765.
0 0 0 2 - 7 8 6138 I9 t 5 l l - 8 e 2 0 $ 0 1 . 5 0 / 0@
owner.
P h o s p h o g l y c e r a t em u t a s e Tc o n v e r t s 3 - P G A t o o - ( + ) - 2 p h o s p h o g l y c e rai cc i d ( 2 - P G A ) : e n o l a s etTh e n f o r m s P E P . C o n s u m p t i o no f P E P b y c o n v e r s i o no f A D P t o A T P . c a t a l y z e db y
pyruvate kinase,Tdrives the overall process.8'e
We havetestedthe PGA methodby usingthe reactionsshown
C T P . G T P , a n d U T P a r e t h e n u c l e o s i d et r i i n S c h e m el l .
p h o s p h a t e ns c e d e di o r t h e s y n t h e s eos f t h e m o s t c o m m o n n u c l e o s i d ep h o s p h a t cs u g a r su s e di n L e l o i r p a t h w a yb i o s y n t h e s e s
pe
h o s p h a t e( r e a c t i o n5 ) i s
( r e a c t i o n sl - - 1 ) . r ' ) D i h y ' d r o x v a c e t o n
u s e d i n r e a c t i o n sc a t a l y z e db y a l d o l a s e s , lal n d a r a b i n o s e5 phosphate(reaction6) is a precursorto 3-deoxy-D-manno-2-oc(KDO-8-P).'2 In all cases,procedures
tulosonicacid 8-phosphate
basedon 3-PGA were as effectiveas and more convenientthan
proceduresbasedon chemicallysynthesizedPEP.5
of CTP from CMP illustratesthe PGA method'r3
The synthesis
of 173g of 3-PGA (barium salt.dihydrate,-957c.,,
A suspension
46 I mmol) in -500m L of water wasstirredvigorouslywith - 600
m L o i i o n - e x c h a n g ree s i n ( D o w e x 5 0 W - X 8 , H + f o r m , 2 0 - 5 0
mesh) ior 30 min at room temperature.The resinwas removed
b y f i l t r a t i o na n d w a s h e dw i t h f o u r 1 0 0 - m L p o r t i o n so f w a t e r . r a
The combined,clear,pale-yellowfiltrateswere neutralizedwith
solid KOH and useddirectly in the next step.
C M P ( f r e ea c i d ,T 1 g , 2 2 0 m m o l ) ,A T P ' N a 2 ' 3 H z O( 1 . 3 3g , 2 . 2 0
m m o l ) ,M g C l , . 6 H 2 0( , 5 1g , 2 5 0 m m o l ) ,a n d t r i e t h a n o l a m i n(e1 . 9
g , l 0 m m o l ) r v e r ca d d e dt o t h e s o l u t i o no f 3 - P G A , a n d t h e p H
was adjustedto pH 7.6 with 5 N KOH. The solution(total volume
(0.25
for 30 min with N2; 2-mercaptoethanol
of I L) wasdegassed
w
e
r
e
a
d
d
e
d
,a n d
t
h
e
n
T
h
e
e
n
z
y
m
e
s
l
5
w
a
s
a
d
d
e
d
.
m
m
o
l
)
mL, 3
(6) From US BiochemicalCorp.:PEP = $4800/mol;3-PGA = $250/mol.
( 7 ) P h o s p h o g l y c e r am
t eu t a s e : R a y , W . J . , J r . ; P e c k ,E ' J ' , J t . l n T h e
E n z y m e s , 3 r de d . ;B o y e r ,P . D . , E d . ; A c a d e m i c :N e w Y o r k , 1 9 7 2 ;V o l . V I '
C h a p t e r 1 2 . M o s t c o m m e r c i a lp r e p a r a t i o n so f 3 - P G A c o n t a i n 2 ' 3 - d i mutase, Enolase:
phosphoglyceric
acid, an activatorof phosphoglycerate
W o l d , F . I b i d . Y o l . V , C h a p t e r1 8 . P y r u v a t ek i n a s e :K a y n e .F . J . l b i d . Y o l .
V I l l A . C h a p t e Ir l .
( 8 ) T h e i s o l a t i o no f P E P i n 1 5 - 2 0 V oy i e l d f r o m 3 - P G A u s i n g e n z y m e
preparations
from yeasthasbeenreported:Ganti,T.; Csoka,A. Magy.,Kem.
: h e m .A b s t . 1 9 7 5 ,8 3 , 1 6 2 1 5 5 v .C s o k a ,A . ; G a n t i ,
F o [ y . 1 9 7 5 ,3 7 , 3 3 , s - 6 C
T . H u n g . T e l j e s8 , 9 3 l ' 1 9 7 4 :C h e m .A b s t r . 1 9 7 5 ,8 2 ' 9 6 4 9 1 c '
(9) The thiee enzymesrequiredare stableunderthe conditionsusedTand
($/ 1000units): pyruvatekinase(0.3,Biozyme);enolase(4.75,
are inexpensive
S i g m a ) lp h o s p h o g t y c e r amt eu t a s e( 4 . 0 0 ,S i g m a ) ;7 0 0 u n i t so f e n z y m ew i l l
convert I mol of reactantsto productsper day under assayconditions.
( 1 0 ) T o o n e ,E . J . ; S i m o n ,E . S . ; B e d n a r s k iM
, . D . ; W h i t e s i d e sG, . M .
Tetrahedron1989.4J. 5635.
, .-D.;
( 1 1 ) B e d n a r s k iM
. . D . ; S i m o n ,E . S . ; B i s c h o f b e r g eNr ,. ; F e s s n e rW
K i m . M . - J . ; L e e s ,W . ; S a i t o ,T . ; W a l d m a n n ,H . ; W h i t e s i d e sG, . M . J . A m .
C'hem.Soc. 1989.t I t ,62'7. Durrwachter,J. R.; Wong, C.-H' "/. Org' Chem.
1 9 8 8 J, J , 4 1 7 5 .
. . ; S i m o n ,E . S . ; S t e i n ,
( 1 2 ) B e d n a r s k iM
, . D . ; C r a n s ,D . C . ; D i c o s i m oR
P . D . ; W h i t e s i d e sG, . M . ; S c h n e i d e r , M .T e t r a h e d r o nL e t t . 1 9 8 8 , 2 9 ' 4 2 7 .
( 1 3 ) C M P i s a v a i l a b l ef r o m M i w o n F o o d s C o . , L t d ' , S e o u l , K o r e a
($200/kg)
(14) The resin was regeneratedwith HCI by following the procedure
by the manufacturer.
recommended
1989 American Chemical Society
8 9 2I
S c h e m eI I . R e a c t i o n sT e s t e db y U s i n g t h e 3 - P G A Method To
RegenerateATP According to Scheme Io
CMP
or
Adenylate klnase
E.C.2.7.4.3
UMP
cDP
or
CTP
Pyruvate kinase 202mmol
E.C.2.7.1.40
----+
or
PEP
UDP
Guanylate kinase
E.C.2.7.4.A
cDP
(1)
UTP
17mmol
Pyruvate kinase
E.C.2.7.1.N
________+
PEP
GTP
'18
mmol
Nucleoside
monophosphate
kinase
E.C.2.7.4.4
llnD
Pyruvate kinasc
E . C . 2 . 7 . 1 . &L
tt?D
(4)
PEP
14mmol
o
Hov,J\/.oH
o
tl
Glycerol kinase
E.C.2.7.1.30
HOv\/OP
17mmol
"$ji"'
HO
Hexokinase
E . C 2. . 7 . 1 . 1
Por
n oH
(--lroH
T-
A f t e r a n a d d i t i o na l 2 4 h . I H N M R s p e c t r o s c o pi vn d i c a t e dt h a t
t h e r e a c t i O nw a s c o m p l e t c . , \ t o t a l o l 1 3 0 m L o f 3 N H C I h a d
b e e nc o n s u m e d .
T o i s o l a t eC T P , - - l - 5 0 - m Lp o r t i o n so f t h e c l e a r .p a l e - y e l l o w
reactionmixture weretransferredinto l2 500-mL polypropylene
c e n t r i f u g et u b e sa n d 1 5 0 m t - o f a b s o l u t ee t h a n o lw a s a d d e dt o
each tubc. .{ r.r,hite
precipitatcformed inrmediatell,'.The tubes
w e r cc o o l e di n a n i c c b a t h l i r r l 5 m i n a n d t h e nc e n t r i f u g e (d4 . C .
1 0 0 0 0 9 l, 0 m i n ) . T ' h ec o r n b r n e ds,t i c k yp e l l e t sw e r ed i s s o l v e idn
6 0 0 m L o f w a t e r .t h e r e s u l t i n gs o l u t i o nw a s d i v i d e da m o n gs i x
2 , 5 0 - n r Lc e n t r i l u g et u b e s .a n d 1 0 0 m L o f a b s o l u t ee t h a n o lw a s
addedto eachtube. The tubesrvereagaincooledand centrifuged.
L y o p h i l i z a t i o no 1 't h c c o n r b i n e dp e l l e t sp r o v i d e d 1 4 , 5g o f a n
o f f - w h i t ep o w d e rc o n t a i n i n g2 0 2 m m o l o f C T P a c c o r d i n gt o e n z v n r a t i ca n a l v s i s i 6 1 9 2 i' i/ e, l d l 9 0 % p u r i t y f o r C T P . K 3 ) . r 7
T h e p r r m a r r l d v r r n t u g er . r tf h e P G A m e t h o da s a r o u t et o P E P
i s t h a t i t r c q u i r c sl e s st i r n c .p r o d u c e sl e s so r g a n i cw a s t e ,a n d i s
moreconvcnrcnt
than rhechcmicals1'nthesis.
lts startingmaterials
a r e c o m m c r c i a l l la v a i l a b l e .I t i s , h o w e v e rm
. o r ee x p e n s i v eT. h e
convenience
outweighsthe differencein cost for reactionscarried
out on a scale( I mol. The convenience
of the PGA method,along
w i t h i t s r e l i a n c co n c o m n t c r c i a l lal v a i l a b l er e a g e n t sr .e c o m m e n d s
i t a s a g e n e r i i tl c c h n i q u ef o r t h e r n s i t u r e g e n e r a t i oonf A T P .
A c k n o w l e d g m e n t\.\ c r h a n k M . J . K i m ( C h i e f o f T r a d e
S e c t i o n ,M i w o n I ' o o d sC . r . .l - t d . .S e o u l ,K o r e a ) f o r p r o v i d i n gu s
with CMP andGMP.
(6)
HO
6 mmol
o A m o u n rt e f e r st o q u a n t i t yi s o l a t e d . =
e ;T p = c \ r i P p h o s p h a tC
dine5'-triphosphate:
UTP = uridine5'-triphosphate;
GTp = guanorin.
5'-triphosphate.
the solutionwas stirredat 22-24 oC under a positivepressureof
N t . A d d i t i o n o f 3 M H C I u s i n ga p H s t a t c o n t r o l l e dr h e p H a t
1.5-7.8.After 48 h, consumption
of HCI had ceased
but rH NMR
spectroscopy
indicatedthat the reactionwas not complete. An
additional32 g of 3-PGA (barium salt, 84 mmol) was converted
to the K* salt as describedand added to the reactionmixture.
( 1 5 )A l l e n z \ m c w
( 1 0 0 0 0u n i t s l.y ' o s e r ct r o n rS i g m a a d e n l l a t kei n a s e
p h i l i z e dp o * d e rf r o meh r c k n
e m u s c l e )p. \ r u v a r ek i n a s e( , 5 0 0 u
0 n i t s l, y o p h i l i z e d
p o w d e rf r o m r a b b i rn r u s c l e )e. n o l a s e( . 1 0 0 0u n i t s .l y o p h i l i z e dp o w d e rl r o m
) e a s )t . a n d p h o s p h o gcler r a t en r u r a s (e5 0 0 0u n i t s s. u s p e n s i oi nn a m m o n i u m
sulfatefrom rabbitmuscle).In thisexample,we usedan excess
of the enzymes
becausetheir cost is low comparedwith the valueof the product.
_- (l0l Keppler,D.ln MethodsoJ'EnzymaticAnalysis,3rd ed.;Bergmeyer,
H . U . , B e r g m e y eJr ., , G r a s s lM
, . . E d s . ; V C H : W e i n h e i ml,9 8 S :V o i V I i . o
, 1 4
+J -.
( 1 7 ) T h e w a t e rc o n t e n(t 1. 1 4 %) w a sd e t e r m i n ebdy t h e K a r l F i s c h em
r et h o d . T h i n - l a v ecr h r o m a t o g r a p hI yp o l v ( e t h y l e n i m i n e ) - c e l l u leolsuea,n t :2 . 0
N H C o o H i I 0 N l - i c l . I : 1 .v T v l i n d i c a t e tdh a t A T p w a sp r e s e nitn a d d i t i o n
t o C T P ; n e i t h e rC M P n o r C D P w a sd e t e c t e dC
. o m p a r i s oonf t h e i H a n d l r p
N-MR-spectra
with thoseof authenticcrp from Sigmaindicatedthe presence
o l - l % e a c ho f , \ T P . d i p v r u v a t e-.I - P G A .i n o r g a n i cp h o s p h a t ea,n de t h a n o l .