Copyright
Reprinted from the Journal of the American Chemical Society, 1985, 107-,7008.
O l98B by the American Chemical Society and reprinted by permission of the copyright
owner.
Glycerol Kinase: SubstrateSpecificityl
Debbie C. Crans and George M. Whitesides*
Contribution from the Department of Chemisty, Haruard Uniuersity,
Cambridge, Massachusetts 02138. ReceiuedMarch I l, 1985
of 28 nitrogen,
catalyzesthe phosphorylation
Abstract: Glycerolkinase(8.C. 2.7.1.30,ATP: glycerol-3-phosphotransferase)
hasdefinedqualitativelythe structuralcharacteristics
sulfur,and alkyl-substituted
analogues
of glycerol. A surveyof 66 analogues
nffissary for acceptabilityasa substrateby this enzyme. This surveywasconductedusingan assaybasedon 3lP NMR spectroscopy
whichdetectedproductsproducedevenby slowreactions.Thesestudiesindicatethat glycerolkinaseacceptsa rangeof substituents
and that the hydrogenatcm atC-2 can be replaced
in placeof oneterminalhydroxylgroup (that which is not phosphorylated),
by other
by a methylgroup. Replacement
of the secondterminalhydroxylgroup (that which is normallyphosphorylated)
of chiral organic
nucleophilic
centersusuallyresultsin lossof activity. Glycerolkinaseis a usefulcatalystfor the synthesis
of kineticconstants
and analogues.Comparisons
substances,
especiallystartingmaterialsfor the preparationof phospholipids
for enzymesfrom four microorganisms(Candida mycoderma,Saccharomycescereuisiae,Escherichiacoli, and Bacillus
productshavestereochemistry
analogousto
stearothermophilus)indicatelittle variationamong them. All phosphorylated
that of sn-glycerol3-phosphate.
Introduction
We and others are developingpractical synthetic methods based
on enzymaticcatalysisfor the selectivefunctionalizationof organic
molecules.2-5 Much of the work concerned with enzymes as
catalysts,both in mechanisticenzymologyand in catalytic organic
synthesis,has centeredon reactionsinvolving naturally occurring
substances.As a result, enzymeshave developedthe reputation
'highly
specific",that is, by implication, of limited utility
of being
for reactionsrequiring transformationsof *unnatural' substrates
(substratesnot encounteredin vivo or not consideredas primary
substratesfor the enzymesof interest). Recent work on enantioselectivetransformationsinvolving esterases(especiallylipase
and hog liver acylaseH) and oxoreductases(especiallyhorse liver
(l) Supportedby the National Institutesof Health, Grant GM 30367.
(2) Whitesides,G. M.; Wong, C.-H. Aldrichimica Acta 1983,16,27-34.
Whitesides,G. M.; Wong, C.-H. Angew. Chem.,Int. Ed. Engl. 1985, 24,
617-638.
(3) Findeis,M.; Whitesides,G. M. Annu. Rep. Med. Chem. 1984, 18,
263-72.
(4) Jones,J. B. In'Asymmetric Synthesis";Morrison,J. O.; Ed.; Academic
Press: New York, 1983.
(5) Battersby,A. R. Chem.Brit. 1984,20,611-6.
has demonstratedthat certain enzymes
alcoholdehydrogenasel0)
can accept a broad range of substratestructuresand still retain
useful in organicsynthesis.
catalytic ratesand enantioselectivities
These enzymes are, however, believed to function in vivo as
broad-spectrumcatalysts,and it is perhaps neither unexpected
nor representativethat they accept a range of substrates. The
work reported in this and the following paperrr was intended to
provide complementary information concerning the substrate
specificity of a representativeenzyme (glycerol kinase) chosen
from the group of enzymesbelieved,on the basisof assignedin
vivo function (reactionsin major metabolic pathways), to have
(6) Ladner, W. E.; Whitesides,G. M. J. Am. Chem. Soc. 1984, 106,
7250-1.
(7) Cambou,B.; Klibanov,A. M. J. Am. Chem.Soc. 1984,106,2687-92.
( 8 ) W a n g ,Y . - F . ; C h e nC
, . - S . ; G i r d a u k aG
s ,. ; S i h ,C . J . J . A m . C h e m .S o c .
1 9 8 4 .t 0 6 . 3 6 9 5 - 6 .
(9) Arita, M.; Adachi,D.; Ito, Y.iSawai, H.;Ohno, M. J. Am. Chem.Soc.
1 9 8 3 .1 0 5 . 4 0 4 9 - 5 5 .
(10) Takemura, T.; Jones,J. B. J. Org. Chem. 1983,48, 791-6, and
referencestherein.
(l l) Crans,D. C.; Whitesides,
G. M., J. Am. Chem.Soc.,followingpaper
in this issue.
0002-7863
@ 1985 American
l8slrs07-7008$01.50/0
Chemical Society
Glycerol Kinase: Substrate Specificity
naruowspecificity.r2 The objectivesof this work were to establish
the range of unnatural substratesacceptedby this enzyme,and
to determine if synthetically useful differencesin substrate selectivity characterizedenzymesderived from different sources.
We selectedglycerolkinase(E.C. 2.7.1.30,ATP: glycerol-3phosphotransferase)for four reasons.
l. Glycerol kinases derived from four microbial sources
(Candida mycoderma, Saccharomycescereuisiae,Escherichia coli,
and,Bacillus stearothermophilus) are commercially available.
This enzyme is inexpensiveand stablewhen immobilized. It has
high specificactivity.
2. The assignedfunction of glycerol kinaser3-enantiospecific
phosphorylationof glycerol and generation of sn-glycerol-3phosphate'a-providesa route to an intermediateimportant in
the synthesisof phospholipids.The ability to synthesizechiral
analoguesof sn-glycerol-3-phosphate
would be useful in the
preparationof new phospholipids.rs
3. Organic phosphatesare relativelyinconvenientto synthesize
using classicalchemicalmethods,especiallyif enantiomerically
enriched materials are required. Synthetic methods basedon
glycerol kinasedo not duplicate existingsyntheticmethods.
4. The supporting technology required to use glycerol
kinase-especiallythat for in situ regenerationof ATP and for
immobilizationof the enzyme-is already fully developed.2.3
The
successfuluse of glycerol kinase in kilogram-scalesynthesisof
sn-glycerol-3-phosphate
and dihydroxyacetonephosphatehas
previouslyestablishedthe practicality of syntheticproceduresbased
on this enzyme.l6-18
Glycerol kinasesfrom C. mycoderma,re-24
E. coli,t2'2s'26
and
B. stearothermophilus2Thave been reported to accept a total of
eight compoundsas substrates.Gancedoet aI.20and Eisenthal
et aI.23have proposedstereochemicalmodels for the active site(s)
of theseenzymes. We have confirmed the results from theseprior
studiesand examineda number of additionalpotentialsubstrates.
Basedon theseexaminations.we were able to define the limits
of syntheticutility of glycerolkinaseand proposea simplemodel
which summarizesthe structuralfeaturesrequiredfor activitl as
a substrate. Comparison of substrateselectivityfor the four
enzymesderivedfrom different sourcesindicatesthat only minor
variationin substrateselectivityis observedand that. with certain
( 1 2 ) T h o r n e r ,J . W . ; P a u l u s ,H . I n " T h e E n z y m e s " B
; o y e r ,P . D . , E d . ;
A c a d e m i cP r e s s :N e w Y o r k , 1 9 7 3 ;V o l . 8 , p p 4 8 7 - 5 0 8 .
( 1 3 ) I n h i g h e ro r g a n i s m tsh e p r i m a r yr o l e o f g l y c e r o kl i n a s ei s t o s a l v a g e
the glycerolreleasedupon lipolysis.In microorganisms
the primary function
of the enzymeis to utilize glycerolas a carbonsource: Lin, E. C. C. Annu.
R e c .B i o c h e m .1 9 7 7 ,4 6 , 7 6 5 - 9 5 .
( 1 4 ) T h e I U P A C c h e m i c a ln a m e f o r t h e c o m p o u n di s n - p r o p a n e - 1 , 2 , 3 t r i o l - l - p h o s p h a t eI.t i s k n o w na s s n - g l y c e r o l - 3 - p h o s p h(aotrel - g l y c e r o l - 3 phosphate)in the biochemicalliterature. The analoguesof sn-glycerol-3phosphate
will be referredto by their systematic
chemicalnamesin this work.
We use oL nomenclaturerather than RS nomenclatureto indicateabsolute
configuration. The namesof the following substratesfor glycerol kinase
i l l u s t r a t eo u r c h o i c eo f n o m e n c l a t u r es y s t e m : D - p r o p a n e - 1 , 2 - d i(o( lR ) propaneI ,2-diol),o-3-fluoropropaneI ,2-diol((S)-3-fluoropropaneI,2-diol),
o - 3 - a m i n o p r o p a n e - I , 2 - d i (o(lR ) - 3 - a m i n o p r o p a n e - I , 2 - d i o la)n, d o - 3 m e r c a p t o p r o p a n1e, 2
- - d i o(l ( S ) - 3 - m e r c a p t o p r o p a n e - 1 , 2 - d i o l ) .
( 1 5 ) R a d h a k r i s h n a nR, . ; R o b s o n ,R . J . ; T a k a g a k i ,Y . ; K h o r a n a ,H . G .
MethodsEnzymol. 1982,72,408-33.
( l6) Rios-Mercadillo,
V. M.; Whitesides,
G. M. "/. Am. Chem.Soc.1979.
t01.5828-9.
( 1 7 ) C r a n s ,D . C . ; K a z l a u s k a sR, . J . ; H i r s c h b e i n B
, . F.; Wong, C.-H.;
Abril, O.; Whitesides,G. M. Methods Enzymol.,submittedfor publication.
( I 8) Wong. C.-H.; Whitesides,G. M. "/. Org. Chem.1983,48, 3199-205.
( l9) Bergmeyer,
H. U.; Holtz, G.; Kauder,E. M.; Mollering,H.; Wieland,
O . B i o c h e mZ. . 1 9 6 1 . J J - i . 4 7 1 - 8 0 .
( 2 0 ) G a n c e d oC
, . ; G a n c e d o J, . M . ; S o l s ,A . E u r . J . B i o c h e m .1 9 6 8 , 5 ,
t65-12.
( 2 1 ) J a n s o nC
, . A . ; C l e l a n dW
, . W . J . B i o l . C h e m .1 9 7 4 , 2 4 9 , 2 5 6 2 - 6 .
( 2 2 )G r u n n e tN
, . ; L u n d q u i s tR, . E u r . J . B i o c h e m . 1 9 6 7 , 3 , 7 8 - 8 4 .
( 2 3 ) E i s e n t h a lR, . ; H a r r i s o n ,R . ; L l o y d ,W . J . ; T a y l o r ,N . F . B i o c h e mJ. .
1 9 7 2 .13 0 , 1 9 9 - 2 0 5 .
( 2 4 ) E i s e n t h a lR, . ; H a r r i s o n R
, . ; L l o y d ,Y l . J . B i o c h e mJ. . 1 9 7 4 , 1 4 1 ,
305-7.
( 2 5 ) T h o r n e r ,J . W . P h . D .D i s s e r t a t i o nH, a r v a r dU n i v e r s i t yC
, ambridge,
M a s s .l 9 7 2 .
( 2 6 ) H a y a s h iS
. . - 1 . ;L i n , E . C . C . - / . B i o l . C h e m . 1 9 6 7 . 2 4 2 ,1 0 3 0 - , 5 .
( 2 7 ) C o m e r ,M . J . ; B r u t o n ,C . J . ; A t k i n s o n ,T . J . A p p l . B i o c h e m . 1 9 7 9 ,
t.259-10.
r985 7009
PEP
ASSAY
i.5h
ASSAY
l_
l=?4h
-to
-20
PPM
rrP NMR spectra
Figurel. Proton-decoupled
of phosphorus-containing
wererecorded
species.
,Allspectra
in deuterium
oxideat pFI7.5.cxcept
whichwasrecorded
thatfor thcphosphoramidatc
at pH 9.5. Thespectra
"
l a b e l e d" a s s a \ \ \ ' e r eo b t a i n e du s i n go t . - 3 - c h l o r o p r o p1a,n2e- -d i oal s
s u b s t r a tfeo r g l r c c r okl r n a suc i t h P E Pa st h ep h o s p h o rdyol n o rf o r t h e
.{TPrege
n c r a l l ( )\n\ s t en r . f h c a s 5 a \ \ ' a sc a r r i e do u t u s i n gt r i e t h a n o l a m i n ea s t h e b u t ' t ' c. r t p l l 5
e x c e p t i o n sk,i n e t i cc h a r a c t e r i s t i cosf t h e s ee n z rm e sa r c s i m i l a r .
We concludefrom thesestudiesthat glycerol kinaseshould be
useful in the preparation of a number of analoguesof snglycerol-3-phosphate,
and that the most useful enzymefor synthetic applicationsis presentlythat from S. cereuisiae.
G l y c e r o lk i n a s ec o n s i s t so f f o u r s i m i l a r o r i d e n t i c a ls u b u n i t s .
T h e e n z y m ef r o m E . c o l i h a s a m o l e c u l a rw e i g h t o f 2 1 7 0 0 0 , 2 6
and that from C. nt)'codermahas a molecularweight of 251000.1e
Glycerolkinasehas beenobtainedin crystallineform from both
E. coli)6 and ('. nr)'t'odernta,re
but to our knowledgeno crystal
structureis available.Gly'cerolkinasecontainssulfhydrylgroups
and is stableonly'in oxi/gen-free
solution.l2Glycerolis essential
for the stability of glycerolkinasein solution.r2Immobilization
of glycerolkinasein PAN gel28markedly increasesits stability;
immobilizedenzyme (from either E. coli or S. cererisiae)lost no
activity on storagefor 6 months at 4 oC, and lost only 30Voof
its activity on use for I month at 25 oC.
High specificactivity of enzymesusedas catalystsin practical-scaleorganicsynthesispermits the constructionof compact
reactors;low cost for the enzymeis critical when poor substrates
are beingtransformedand when largequantitiesof enzymemust
be employed. Glycerol kinasesfrom the four commercialsources
examinedhavespecificactivitiesof 50-100 u/mg (measuredwith
glycerolas substrate)and cost$50-125 per 1000U (l U = I prmol
of product formed per min; 700 U produces- I mol of product
per day).
Results
QualitativeStudiesof SubstrateReactivity. Previousstudies
of the substratespecificityof glycerolkinasehave relied on en( 2 8 ) P o l l a kA
. . ; B l u m e n f e l dH,. ; W a x , M . ; B a u g h nR
. . L . : W h i t e s i d eG
s ..
M . J . A m . C h e m .S o c .1 9 8 0 .1 0 2 . 6 3 2 4 - 3 6 .
(-rans and Whitesides
7010 J. Am. Chem. Soc., Vol. 107, No. 24, 1985
zymatic assays.le-27
Enzymatic assaysare unreliablefor poor
substratesbecausetheseassaysmay not function reliably in the
concentratedsolutionsof reactantsrequired to achievemeasurable
rates,and becausethey do not distinguisha slow reactiondue to
a poor substratefrom a slow reactiondue to low concentrations
of reactiveimpurities in a nonsubstrate.In this work we have
relied on a semiquantitativeprocedure based on rrP NMR
spectroscopyto follow the rates of phosphorylationof substrates
and to identify the structuresof the phosphorylatedproducts.
PhosphorusNMR spectroscopyis particularly well suited to follow
enzyme-catalyzedphosphorylationreactionsfor severalreasons.
First, enzymaticactivity in phosphorylationof substrateis clearly
differentiated from ATPase activity. Second,the severaltypes
of phosphoruscompoundsof interestin thesestudies(nucleoside
(PEP),
phosphates,inorganic phosphate,phosphoenolpyruvate
organicphosphates,
amidates,and thiophosphates)
havedistinct
and characteristicchemicalshifts.2e Third, 3lP NMR assaysare
applicableto studiesin the concentratedsolutionsof interestin
organic synthesisbecausesmall concentrationsof contaminating
reactivesubstratesare easily identified. TheseNMR assayscan
also be carried out at high or low valuesof pH. Fourth, other
typesof spectroscopic
information(phosphorus-proton
coupling
c o n s t a n t sc,h a n g e si n c h e m i c a sl h i f t w i t h c h a n g e si n p H ) c a n b e
h e l p f u li n a s s i g n i n gs t r u c t u r e .T h e c e n t r a la d v a n t a g eo f t h e r i P
N M R a s s a yi s , h o w e v e rt.h a t i t u n a m b i g u o u s l fl o l l o u s t h c f o r m a t i o n o f t h e p r o d u c ta n d i s n o t m i s l e db 1 c o m p e t i n gs i d e r e actions.
Figure I summarizesllP NMR spectraof a number of compoundsimportant in theseassays,togetherwith spectraobserved
during one representativeassayprocedure. We emphasizethat
the resultsobtainedusing this assayprocedureare qualitative;
it is possibleto differentiatebetweensubstratesthat react rapidly,
those that react slowly, and those that do not react under the
conditionsemployed. Quantitative comparisonof rates depends
upon classicalkinetic techniquesreportedin a subsequentsection.
Table I summarizesthe resultsof thesesurveyexperimentscarried
out by I'P NMR spectroscopy.Triethanolaminewas used as
buffer at pH 7-8;glycine was usedat pH 9-10.30 All potential
substratescontainingnitrogenwere examinedboth at pH 7.5 and
pH 9.5-10 in order to test for reactivityof protonated(RNH3+)
and unprotonated(RNH2) materials. Certain of the substrates
listedin this table which show little or no activity were relativelv
i m p u r e( i m p u r i t y > 9 0 % , ) .W e b e l i e v et h a t t h e l o w a c t i v i t i e sa r e
r e a l ,b u t w e n o t e e x p l i c i t l yt'h a t t h e y m i g h t , i n p r i n c i p l e ,r e f l e c t
inhibition of the enzyme by impurities.
The entriesin Table I are classifiedrelative to the structure
and reactivityof glycerol(eq I and 2). The structuralfeatures
YZ
\ (^/
,/"
ArP
----+
\
R
GK
CH XH
2
HOH
\ 'c/
(l)
I
,zc\
R
cHzx
P(o-L
HOH
ArP
\.t
'-'----------GK
,/"\
R
YZ
\"'
CH2OH
,/"\
R
e)
I
c H 2O P ( O - ) z
listed in the table are those that differ from thoseof glycerol. An
entry r = ***
indicatesthat the reactivity of the indicated
substanceis > 10% that of glycerol;r = ** indicatesthat the
reactivity is llVo ) r ) 0.17othat of glycerol; r = * indicatesthat
the reactivity is <0.lVo that of glycerol but still detectable. A
reactivity of 0 indicatesthat no detectablereaction occurred over
72h. The productsincludedunder the group r = * were characterizedonly by 3rP NMR spectroscopyand were not isolated
( 2 9 ) M a r k , V . ; D u n g a n ,C . H . ; C r u t c h f i e l d ,M . M . ; V a n W a z e r ,J . R . I n
*Topics
in PhosphorusChemistry";Grayson,M., Griffith, E. J., Eds.; IntersciencePublishers:New York, 1967;Vol. 5, pp 227-457.
(30) KtihneH
, . ; L e h m a n ,H . - A . ; T o p e l m a n nW, . Z . C h e m . 1 9 1 61, 6 , 2 3 - 4 .
o r o t h e r w i s ei d e n t i f i e d .A l l o f t h e c o m p o u n d sl i s t e di n T a b l e I ,
e ,-2 - d i o l , 2ar n d
e x c e p t D - p r o p a n eI -, 2 - d i o l , 2 o
3 - 3 - f l u o r o p r o p a n1
were examinedin this work; certainof
2-fluropropane-1,2-diol,23
t h e m h a v e a l s o b e e n e x a m i n e dp r e v i o u s l yb y o t h e r s . l e2 7 T h e
r e s u l t so f o u r s t u d i e sa n d t h e s ep r e v i o u sr e p o r t sa r e i n g o o d
q u a l i t a t i v ea g r e e m e n t .
The most importantconclusionfrom Table I is that a number
(28) of compoundsother than glyceroland dihydroxyacetone
are
substratesfor glycerolkinases.Becausethe enzymeis inexpensive.
evenrelativelyunreactivesubstrates(r = ++) can be considered
f o r p r a c t i c a l - s c a l(e> 1 0 g ) p r e p a r a t i o n su s i n g t h i s e n z y m e . A
second observationis that the phosphorylationof substrates
containingnucleophilicsubstituents(nitrogenand sulfur) is not
y f t h e s u b s t i t u e n t .D t . - 3 d i c t a t e ds o l e l yb y t h e n u c l e o p h i l i c i t o
on nitrogenevenat pH
is phosphorylated
Aminopropane-1,2-diol
7 . 5 , a t w h i c h p H 9 9 V oo f t h e a m i n o g r o u p sa r e p r o t o n a t e d . 3 l
1,2-diolis phosphorylated
on the hydroxyl
ot--3-Mercaptopropanegroup evenat pH 10.5,at which pH 9O%of the thiol groupsare
deprotonated.3lA third observationconcernsthe greaterflexibility
in substratestructuresthat are acceptedby the glycerolkinase
from S. cereri.riaerelativeto that from E. coli. Both enzymes
s h o w h i g h s u b s t r a t ea c t i v i t y w i t h g l y c e r o l , o t - - 3 - m e r c a p t o p r o p a n eI-, 2 - d i o l .a n d o l - 3 - m e t h o x y p r o p a n1e,-2 - d i o l .T h e i n a c tivity (r = 0) of glycerol kinase from E. coli with compounds
having R groupslarger than CH2OCH, contrastswith the greater
toleranceof the glycerolkinasefrom S. ceret,isiaefor substituents
in this position. The S. cereuisiaeenzyme acceptsDL-3-thio1,2-diol
1,2-diol(r = +++), ol-3-ethoxypropanemethylpropane(r = **). ol-butane(r = **), or--3-thioethylpropane-1,2-diol
1 , 2 - d i o (l r = * * ) , a n d o l - 1 V - a c e t y l - 3 - a m i n o p r o p a n e - 1 , 2 (- rd i o l
= +). The glycerolkinasefrom E. coli does,however,selectively
pr--butanephosphorylate
1,2,3-triolat C- I , while the enzymefrom
S. cereuisiaeappearsto be lessselective. A fourth observation
concernsthe greater flexibility in substratestructuresat C-2
acceptedby the glycerol kinase from C. mycodermc relative to
that of E. coli and S. cereuisiae. Substrate activity of nl-2with glycerol kinasefrom C. mycoderma
methylpropane-L,2,3-triol
has previouslybeen reported2a(and confirmed by our 3rP NMR
assay). We observedno substrateactivity with glycerol kinase
from E. coli and S. cereuisiae.
Certain of the observationsin this table require brief explan a t i o n . F i r s t .a r a c e m i cm i x t u r e o f p r o p a n e - l , 2 - d i oi ls i n a c t i v e
even though the pure D enantiomeris phosphorylated.23The
inactivity of the racemic material reflects the weak substrate
activity of the o enantiomerand the stronginhibitory capability
of the L enantiomer.23The substrateactivity (r = **) of or-butane-I ,2-diolcontrastswith the inactivityof ot--propane1,2-diol
with glycerol kinasefrom S. cereuisiae,and probably reflectsthe
fact that L-butane-1,2-dioldoesnot bind as stronglyto the enzyme
as does L-propane-1,2-diol.Activities reported for dihydroxyacetoneand derivativesrequire caution in interpretation: dihydroxyacetoneexists in aqueoussolution primarily as its hydrate;33a-hydroxy ketonesare also hydrated. The actual substrate
for the enzymatic reaction is probably the hydrated form of these
compounds,20
although this mechanistichypothesishas not been
proven experimentally.
A number of other substancesnot fitting convenientlyinto the
organization of Table I were also establishednot to act as substrates for glycerol kinase: cis- and trans-cyclohexane-1,2-diol,
cis- and trans-cyclohexane1,3-diol,2,2-bis(hydroxymethyl)l-
(31) The pK" valuesfor protonatedol-3-aminopropane-1,2-diol
and for
were determinedfrom their titration curves
ot--3-mercaptopropane-1,2-diol
to be respectively9.4 and 9.6.
(32) Eisenthalet a1.23determinedthe K. for l-propane-1,2-diol,r--3fluoropropane-1,2-diol,
and 2-fluoropropane-1,3-diol
to be respectively
45, 165,
and 100 mM. They also determinedK, for o-propane-1,2-diol
and o-3fluoropropane-1,2-diol
usingglycerolas substrateto be respectively
4.6 and
8.6 mM (usingdihydroxyacetone
as substrate,the K, valuesare 1.2and 3.6
mM). Their work was carriedout usingglycerolkinasefrom C. mycoderma.
We confirmedwith the 3'P NMR assaythat glycerolkinaselrom S. cereuisiae
and f. coli catalyzed the phosphorylationof glycerol in the presenceof
o r - p r o p a n e1-, 2 - d i o(l l 0 m M ) .
(33) Befl, R. P. Adu. Phys. Org. Chem. 1966,4, l-29.
J Am Chem. Soc.' Vol. 107' No.24, 1985 7011
Glycerol Kinase: Substrate Specificity
TableI. QualitativeSubstrateActivitiesof CompoundsHavingthe StructurexCHICYZR (Eq l) for Glycerol Kinasefrom s. celerlrlae
a ct i v i t y
OH
cH2oH
H
CH,
CH,F
cH,cl
cH, Br
CH]SH
cH2NH2
cHzocHs
cH,ococH.
cHrocH2cH3
CH,SCH3
cH2scH2cH3
CH,NHCHs
cHrN(CH3)2
cH,NHCOCH3
CH=O
cH(ocH3)'
COOII
cFI,CI{3
c H r c l { ro H
( . H ( C H)3O H
c H O H C F IO
TI I
CH,CN
cHrcH2cH3
OH
(:O)
(:O)
(:O)
H
CH,
cH2cH2oH
H
H
CH.,
(=o)
(ocH,)
H
H
H
H
H
H
H
H
H
H
H
cH2 oH
cHroH
NH,
NH,
NHt
NH,
NH,
NH,
H
A-fPascb
(D onlv)c
{D onlv)c
+r
24
i
T
TT
+++(0)e
+ + +( 0 ) e
+++(0)e
+ + (0)€
gd
0d
+ (0)€,/
-rr
gh
r
-r + (0)e
(D ATPase)c
A T P a s eb
A T P a s eb
0
+ t-r
0
0
+
0
0
0d
H
CH,SH
FI
CH,
It
CH,
cH2NH2
cH,cl
c H ,ocH .
c H (cH .)oH
1^
L1
--(--)€
( - H rN H 2
CH,CX-H.CII.
C I I . ( - II .
CH.
CH,CH3
cHroH
CH,CH3
iB,l9
{l
i 8 . 1 9 .2 1
,\ I'Purc f
ATPasc/
I 8 , 1 9 ,2 1
TT
ct{(cHr)oH
CH,C}I,CHJ
2I
2I
19,33
Tta
0
cr{.cH,o}{
t 7- 2 4
T ' -
cHrcl
C I I .B r
I]
SH
SH
NHt
NHt
NHt
+++
++
+
+
+ - ++
ref
notcs
(l
{l
il
(l
D.L rll tt
0
6i
0t
0
0
j
+-1
0
0
pd
)2
21
gd
6d
D.L all 0
.' td
t)d
od
D,Dr, all 0
11d
gd
gd
r\TPasef
0
a Thc structuralfeatureslisted are thosc that drtar from glycerol. Unlessindicatedotherwise,thc astivitieswereindistinloishableusingth€
g l \ e c r okl i n a s ef sr o m , Sc. e r c f i s i aaen dI,: . . o / i . A n e n t r y r = + + + i n d i c r t c s t h a t t h e r e a c t i v i t y o f t h c s u b s t a n c e i s r > l 0 T t h a t o f g l y c e r o l ;
| = ++ indicatcsthnt the reactivityis l0% > / > 0.1%that ofglycerol;/= + indicatcsthrl the rcactivityisr < 0.t% that ofdycerol or thrt the
r = 0 indi_
rc clivity is accompanicd
infornrutionon spccificsubstrates;
by significant
sidereactions.Seefootnotes.,/,o.te\tfor det.riled
tri
cltcs that no dctectirble
rcactionoccurredovcr 72 h. Unlcssindioatedothcrwise,thc reactions
wcrccardcdout undertheseconditions;
'C),
(250mM). The
cthanolamine
chloride(12.5mM), PEP(65 mM). ATP (6.5 mM). and substrate
buffer(0.J M, pH 7.5.30
mirgnesirm
$as accompanied
by ATPaseactivity(<407 of
solutioncontainedDzOasintefn,rlNMR lock. o Thc activityin subslrdte
Fhosphorylation
(L) is sucha potentinhibitorthat only the pureD enrntiomcris phosphorylated.Racemic
lotal nctivity). c Thc unrcnctive
enantionrer
rrri\trfrcs
do not showsubstrrteilctivity. Thescstudieswerecarriedout usingglyccrolkinascfrom C n4.coderna.1tThe in ctivity of r!cenic proprnc-1,2-diolwrs confirmcd for glyccrol kinasctrom both ,s.reretisia?,tnd l.:...r/i. d The relcdons were carriedout at pH 7.5 (deseribcdin footnote./).rndnH 9.5 in orderto e\arninethe substratcclivity ofboth the prolonatedand nonprolonated
forms. Al pH 9.5 thc
(250
fcrctkrnsolutioncon![ncd thc following: glycinc(0.3 M), malnesiumchloride(12.5nrM).PI]P(65 nrM).ATP (6.5 mM), and substrare
nrM). e Thc .rctivit! ()f thc indiciltcd substanccwaslbund to bc diffcrcnt with dyccrol kin selrom ^!'.@ret'isiaea')d[:. coll. The rcactivily
irdiculedin plrcnthcscs
sidereilction(>407 of totnlirctivity).
is that with !lyccrol kinaseliom Z. col1. / tlvdrolysisof ATP wrs a rLrbstantial
g
ob_
Ihc ATPrscactivitynlay be induccdby rhc substance
or n1ir!,
rcflcctr.rpidhydrolysisof rciction products, Only ATPIscitclivity\,_us
I 2-Methylpropanc\ervcd. n The irclivitvwitsdcterminedin the prcscncc
ol hydf.lzine(0.4 M) in ordcr1oremovcresidul D-glyccraldehyde.
bul nol \r'ith!:l)cerolkin sesfroD I;.
kinlscitonr(.'It1!0|l(|1)]d'Th!'ilctivi!yol.1hisfon]p{}tInd$lsn.)te\:ln]incdwith!ltccrolkinnseI()In,/:
Crans and Whitesides
7012 J. Am. Chem. Soc.. Vol. 107, No. 24, 1985
I
tl
ll
75 70
I
. \,J..
-
_
rlrrrrlrrrrl
75
I
70
d
ll
,*,* **-{|Jl,**l lt,-,* .-,*,., *I
'r
l,
J-_^-_..-=_J*
trlrrtltr
7570--LrJ--
c
'1ty1,1-.,^a-f-sf
.tr^-t'-r-zlff+----r.*ag
loo
o
PPM
Figure 2. Proton-decoupledr3C NMR spectra of o-3-chloropropane1,2-diol-I -phosphateand related species: (a) nr--3-chloropropane1,2diol, (b) n-3-chloropropane-1,2-diol-I -phosphateprepared by phosphorylation catalyzed by glycerol kinase, (c) authentic or--3-chlorop r o p a n e - 1 , 2 - d i oll--p h o s p h a t e ,( d ) o f f - r e s o n a n c e
d e c o u p l e ds p e c t r u mo f
( b ) , ( e ) a u t h e n t i cs n - g l y c e r o l - 3 - p h o s p h a t(ef ), a u t h e n t i c o r - g l y ' c i d o l - -l
phosphate.
h
//,
4'*ll
rlrrll
76.8
76.5
o*'_o'.:l-l*l
_
ii
ll
ll
'"'-l l*"
77
76
ll
ll
o
*"**-**JUL*.J{*******
-f-*,-r*
1oo
PPM
o
Figure 3. Proton-decoupledr3C NMR spectra of n-3-aminopropane1,2-diol-3-phosphaa
t en d r e l a t e d s p e c i e s : ( a ) n l - 3 - a m i n o p r o p a n e - 1 , 2 d i o l , ( b ) o - 3 - a m i n o p r o p a n e1-, 2 - d i o l - 3 - p h o s p h a pt er e p a r e db y p h o s p h o rylation catalyzedby glycerol kinase, (c) ur--3-aminopropane1,2-dioll-phosphate prepared chemically (eq 4), (d) off-resonancedecoupled
s p e c t r u mo f ( b ) , ( e ) l - 3 - a m i n o p r o p a n e - 1 , 2 - d i o l - l - p h o s p h apt e
repared
c h e m i c a l l y( e q 5 )
butanol (2-ethyl-2-hydroxymethylpropane1,3-diol), D-ribose,
o-arabinose, DL-3-mercaptopropane-1,2-diol disulfide, and or--Sacetyl-3-mercaptopropane-1,2-diol.
or--S-Acetyl-3-mercaptopropane-1,2-diol was not recovered after the reaction; its inactivity
may reflect acetyl migration from sulfur to oxygen under the
reaction conditions.
3rpNMR .J;1"Figure4. c:pu.ison of oro,on-o.loupled
or the
phosphoramidate
andthephosphate
esterof or--3-aminopropane1,2-diol:
(a) n-3-aminopropane(b) o-3-aminopropane1.2-diol-3-phosphate,
1,2(c) o-3diol-3-phosphate
and o-3-aminopropane1,2-diolI -phosphate,
aminopropane1.2-diolI -phosphate.
Identification of the Structures of Phosphorylatedhoducts. For
many of the substrateslistedin Table [, it was possibleto establish
the structuresof the phosphorylatedproductsdirectly by examination of lrP NMR spectra. For others,more detailedexamination was required. In many cases,eventhough the structure
of a product could be deduced directly from knowledge of the
structure of the substrate and from the 3lP spectra, the phosphorylated product was isolatedand examinedusing other techniques. Carbon-13 NMR spectroscopyproved particularly useful
in this context. Specific characterizationsfollow.
The isolationand identificationof the phosphorylatedproduct
from ol-3-chloropropane-1,2-diol
are of particular interest,since
this substanceis currently in use as an antifertility agent for
rats.l4'3s The activity of this compound as a substratefor glycerol
kinase has previously been ascribed to glycerol impurities.36
Glycerol is presentas an impurity in or--3-chloropropane-1,2-diol,36
and it is also formed in aqueoussolutionsunder basicconditions,36
presumablyvia glycidol as an intermediate. It is thereforedifficult
to distinguishwhetheractivity detectedby enzymaticassayis due
to ot--3-chloropropane-1,2-diol
or to the product of hydrolysis.
Figure 2 summarizesrelevant '3C NMR spectra. The assignment
of the structure 3-chloropropane-1,2-diol-l-phosphate
to the
phosphorylatedproduct restson two piecesof information. First,
the r3C NMR spectrumis indistinguishablefrom that of authentic
ot--3-chloropropane-1,2-diol-l-phosphate.37
Second,both r3Cand
3rP spectra of this material are clearly distinct from those of
(or or,-glycerol-3-phosphate)
authentic sn-glycerol-3-phosphate
and ol-glycidol-l -phosphate.
The compound isolated following phosphorylation of or--3aminopropane-1,2-diol
at pH 10.0(eq 3) was assignedthe unusual
HOH
ATBGK
\/
Ho.=X=_ruH,
#;-
HOH
O
Ho_,LNHP(o-L (3)
75 "/"
structure of the correspondingphosphoramidatebased on five
piecesof evidence.First, the 3lP andl3C spectra(Figures3 and
4) of this material were indistinguishablefrom thoseof authentic
(34) Lobl, T. J. Clin. Androl.1980, J, 109-22.
( 3 5 ) J o n e sA
, . R . A u s t . J . B i o l . S c i . 1 9 8 3 ,- t 6 ,3 3 3 - 5 0 .
(36) Brooks,D. E. ,/. Reprod.Fertil. 1979,56,593-9.
(31) Zetzsche,F.; Aeschlimann,F. Helu. Chim. Acta 1926.9.708-14.
107, No. 24, 1 9 8 5 7 0 13
Glt'cerol Kinase: Substate Specificity
racemic material (eq 4).18 Second,the observationof a 2,/pNc
Ho H
\/
l ) P o c l 3P'Y
Ho H
cHacN,o"c
a
o
,l
\/
t.
H O_ v _ N H P ( O - ) @
2 l
HO_V_NHz
;ffi
do*
I
b
= 1 . 6 H z ( + 0 . 3 ) f o r t h e c a r b o na t t a c h e dt o t h e n i t r o g e ni n t h e
rrC NMR spectrum is compatible with N-phosphorylation.3e
Third. this substanceis stablein basicsolutionand decomposes
rapidly in acidic solution.ao No decompositionwas observedfor
chemicallypreparedot--3-aminopropane1,2-diol-I -phosphateafter
2 weeksat ambient temperatureat pH ( I . Phosphoramidates
are basestableand acid labile.ar Fourth, the 3rPand r3CNMR
s p e c t r a o f o - 3 - a m i n o p r o p a n e1-, 2 - d i o l I- - p h o s p h a t ep r e p a r e d
chemically (eq 5) are distinct from thoseof the product from the
HOH o
NH3,Hzo
-fr?
oilro-r,
c,---h-,
Ho H
,t
73
NHz
--\.-oi,to-r, (5)
( 3 8 ) H o r n e r ,L . ; G e h r i n g ,R . P h o s p h o r u S
s u l f u r 1 9 8 1 ,1l . 1 5 1 - 7 6 .
(39) Under these conditionsno coupling was observedfor the carbon
a t t a c h e dt o t h e p r i m a r y h y d r o x y lg r o u p .
( 40) The half-livesfor nl- 3-aminopropane1,2-diol3-phosphate
at various
, l ; 1 0 . 0 , 3 3 61; 1 . 0 , 9 6 0 ;
p H a r ea s f o l l o w s( p H , l ' 7 2 . 6 )7: . 0 , 5 ;8 . 0 ,1 8 ; 9 . 0 5
12.0>
. 5000.
( 4 1 ) F l u c k ,E . ; H a u b o l d ,W . I n " O r g a n i cP h o s p h o r uCs o m p o u n d s "K; o s o l a p o f fG
, . M . , M a i e r , L . E d s . ;W i l e y - l n t e r s c i e n c eN:e w Y o r k , 1 9 7 3 ;V o l .
6 . C h a p t e r1 6 ,p p 5 8 0 - 8 3 1 .
( 4 2 ) M c F a r l a n e ,W . ; P r o c . R . S o c .L o n d o n ,S e r .A 1 9 6 8 , 3 0 6 ,1 8 5 - 9 9 .
, . E . ; R a n d a l lE
. . W . ; B r i t t o n ,H . G . ; F a z a k e r l e y ,
CurzonE
. . H . ; H a w k e sG
the coupling
G. V. J. Chem.Soc.,Perkin Trans.2 1981.494-9. For reference,
= 5.-5,
c o n s t a n t sf o r . r n - g l y c e r o l - 3 - p h o s p haartee r e s p e c t i v e l(yH z ) : : . . / 6 e p
t " / . . o p= 7 . 3 ( p H - l ) : 2 J c o p = 3 ' 5 , r " / ( ' ( ' o=p 6 . 2 ( p H - 1 2 ) '
l,
70
,*...,,J*^-*,,ir**
o
phosphorylationcatalyzedby glycerolkinase(Figures3 and 4).
F i f t h , t h e o - 3 - a m i n o p r o p a n e - 1 , 2 - d i o l - 1 - p h o s p hpart e p a r e d
chemicallydoes not transfer a phosphorylgroup to nitrogen to
form o-3-aminopropane1,2-diol-3-phosphate
in aqueoussolutions
(pH l-14, ambienttemperature).The phosphoramidate
isolated
from the enzymaticreactionis thus not formed by rearrangement
of an initially formed phosphateester.
Phosphorylationof ot--3-mercaptopropane1,2-diolwas confirmed to be on the C-l hydroxyl group by ''C NMR spectroscopy:
in particular,the two-bondP-C couplingconstantwas that expected('"/poc = 3.J H.).0' In addition, this substanceoxidized
to the correspondingdisulfide on standing overnight in solution
at pH l3 in contact with air. Addition of sodium borohydride
to a solution of the disulfide regeneratedthe thiol quantitatively
(as observedby both r3C and 3rP NMR spectroscopy).
The siteof phosphorylation
of or--butane1,2.4-triolwas assigned
a s C - l o n t h e b a s i so f i t s l 3 C s p e c t r a( F i g u r e 5 ) . T h e m o s t
important observationis that of a three-bondP{ coupling to the
carbon atom bearing the secondaryhydroxyl group expectedfor
phosphorylationat C-1 ('/pocc = 9.2 Hz).
The site of phosphorylationof ot -butane-I,2,3-triol was assigned
a triplet is
to the C- I hydroxyl on the basisof 3rPspectroscopy;
observedin the proton-coupled3rP NMR spectrum. The l3C
NMR spectraconfirmedthat the phosphorylationtook placeat
C- l; in particular, the two-bond P-C coupling constantwas that
expected(t/ooc = 3.7 Hz).
The assignmentof structuresto other compoundslisted in Table
I is basedon similar, although lesscomplex,3rPand l3C NMR
data. Thesedata are summarizedin the ExperimentalSection
( T a b l eI V ) .
Assignmentof AbsoluteConfiguration. Assignment of absolute
configurationto the products obtained from phosphorylations
catalyzedby glycerolkinasewas basedon activity of thesesub(8.C.
dehydrogenase
stancesas substratesfor glycerol-3-phosphate
. is
1 . 1 . 1 . 8s. n - g l y c e r o l - 3 - p h o s p h aNt eA:D 2 - o x i d o r e d u c t a s eT) h
oxidation of sn-glycerolassavdependsupon the stereospecific
a n d s t r u c t u r a la n a l o g u e s( o - 3 - f l u o r o p r o p a n e - 1 , 2 3-phosphate
diol- I -phosphate)to dihydroxyacetonephosphate (and analogues).al'44The lack of substrateactivity of the enantiomer
r
1oo
o
ppM
F i g u r e 5 . P r o t o n - d e c o u p l e rdr C N M R s p e c t r ao f o - b u t a n e - 1 , 2 . 4 - t r i o l | - p h o s p h a t ea n d r e l a t e ds p e c i e s :( a ) o l - b u t a n e -1 . 2 , 4 - t r i o l(. b ) o f f - r e s o n a n c es p e c t r u mo f ( a ) , ( c ) o - b u t a n e I- , 2 , 4 - t r i o l -I - p h o s p h a t ep r e p a r e db y
decoupled
phosphorvlationcatalyzedby glycerolkinase,(d) off-resonance
s p e c t r u mo f - ( c ) .
T a b l e I I . L s t i n r a t c sr r i [ - - n a n t i o n r e rai cn d C h e m i c a lP u r i t i e so f t h e
T P X C H T C Y Z Rc.q I )
rtL
. \ n a l o g u co
s l ' r r r - ( i l r r , c r u l - . 1 - p h t r s p( hTi O
P r e p a r e db y C i r e c r r ) l K l n a \ c ( a t a l rz c d P h o s p h o rl ra.t i o n a s
a tcch r d r o g c n a s(cG D H . e q 6 ) d
* r t h ( j i r c c r t r l - - i - p h o s p hD
Determined
'rP
\ \1R Spectrorcopr^
and Quantitativc
a ssay'
GDH(%,)" rrP NMR(%)b
OH
OH
cH20H
cH2cl
CH2Br
cH:ocH,
c'HrsH
\H.
( ' l J : ( ' H :)(l l
>97
>97
95
94'
9-5d'
>91
I
>95
>95
>95
>95.
>95"
>95
>95
" T h c en r i l n ; ; t ;
n..,t^ rl ,I*'
b y e n z y n r a t i ca s s a \u s r n gs n - g l rc e r o l - - 1 - p h o s p h da ct ch r d r o g e n a s eT. h e
l e c t i o n . ^T h e
a s s a y c o n d i t i o n sa r e d e s c r i b e di n t h c L . r p e r i m e n t aS
c h e m i c a l p u r i t y o f t h e o r g a n i c p h o s p h a t e sw a s d e t er m i n e d b 1 ' q u a n t i t a t i v e r r P N M R . S o d i u m p h o s p h a t ew a s u s e da s t h e i n t e r n a l s t a n d a r d .
T h e r e c o r d i n gp a r a m e t e r sa r e d e s c r i b e di n t h e E , x p e r i m e n t aSl e c t i o n .
' l n o r g a n i c p h o s p h a t e( 4 % ) w a s o b s e r v e db y q u a n t i t a t i v el r P N M R .
d D i t h i o t h r e i t o l( D T T , I m M ) w a s a d d e d t o t h e a s s a y . " l n o r g a n i c
p h o s p h a t e( 2 % \ w a s o b s e r v e db y q u a n t i t a t i v e3 r P N M R . / o - B u t a n e 1 . 2 , 4 - t r i o lI-- p h o s p h a t ew a s n o t a s u b s t r a t ef o r . r n - g l y c e r o l - 3 - p h o s p h a t e
dehydrogenase
having thc opposite cont'rgurationat C-2 is well documented.a3'45
T h e a s s a r i s c a r r i c d o u t i n t h e p r c s e n c eo f h y d r a z i n e i n o r d e r t o
d r i v e t h e c q u i l i b r i u m 1 e q6 ) . r "
H0 .H
O
;
t.'
HO _* _., OP(O-L
G D HN
, AD
. --r _, *
22
H,
,N
N
O
i
tt
HO _ u _ Op(O-)
(6)
Our results are summarized in Table IL The close agreement
b e t w e e n e s t i m a t e so f e n a n t i o m e r i c a n d c h e m i c a l p u r i t i e s o f t h e
s a m p l e s i n d i c a t e s h i g h e n a n t i o s e l e c t i v i t yf o r t h e r e a c t i o n s . F o r
e x a m p l e , f o r 3 - c h l o r o p r o p a n e -1 , 2 - d i o l -I - p h o s p h a t e , ) 9 7 % o f t h e
and is, as a result, assigned
sample was oxidized by NAD/GDH,
t h e s t r u c t u r e D - 3 - c h l o r o p r o p a n e - I , 2 - d i o l - I - p h o s p h a t e 1)' 9 5 7 o o f
t h e s a m p l e c o n t a i n e d ,b y ' ' P N M R , 3 - c h l o r o p r o p a n e - 1 , 2 - d i o l - l ( 4 3 ) M i c h a l ,G . ; l - a n g G
c n a l y s i s " . 2 nedd . :
, . I n " M e t h o d so f E n z y m a t i A
, 9 7 4 ;V o l . 3 , p p l 4 l 5 - 8 .
BergmeyeH
r , . L i . ,E d . ;V e r l a gC h e m i e :W e i n h e i m 1
( 4 4 ) G h a n g a sG
. . C . ; F o n d y ,T . P . B i o c h e m i s t r y1'9 7 1 ,1 0 , 3 2 0 4 - 1 0 .
( 4 5 ) F o n d y ,T . P . : P e r o ,R . W . ; K a r k e r ,K . L . ; G h a n g a sG
, . S.; Batzold,
F . H . J . M e d . C h e m .1 9 7 4 .1 7 . 6 9 ' 7 - 7 0 2 .
( 4 6 ) F o n d y ,T . P . ; G h a n g a sG
, . S . ; R e z a ,M . J . B i o c h e m i s t y 1 9 7 0 , 9 ,
3272-80.
7014 J. Am. Chem. Soc.. Vol. 107. Nct.24. 1985
Crans and Whitesides
Trble IIl, ApparentValuesof K. and RelativeValuesof Z** for Phosphorylation
of CompoundsHaving the StructureXCHTCYHR Catalyzed
by GlycerolKinaseo
C. myco
K. (mM)
K. (mM)
B. stearo.
E. coli
S. cere
v^ *b (Vo)
K. (mM)
V^^^'(vn)
K. (mM)
V^^*D(Vo)
V^u^b(7o)
100
0.054
r00
I07'
12
1.2
cH2cl
t2.9
r03
87
't4
74
CH2Br
t2.1
3.4
87
cH2NH3* d
2.2
43
65
1.4
38
cH2NH2"
30
z. -)
CH25H
t.2
97
1.4
l0l
2.1
95
il0
cH2ocH3
1.7
104
4.0
95
2.9
104
1.5
92
21
0.5
80
86
0.5
51
I
1.6
cH2cH20H
2
0.6
6.4
H
0.04
l8
'Reaclions w€re carried out under theseconditionsunlessindicatedotherwise: pH 7.6, 25 oC.
[ATP] = 5 mM, [Mg'?+]= 15.6 mM, [tri= 50 mM, INADH] = 0.16mM, [K+] = 1.03mM, pyruvatckinasc= 6 U/mL, and lactatedehydrogenase
= 12 U/mL. Boththe
ethanolaminel
(m and ,/sii valuesare in mo6tcasesreproduciblewithin | 0%. In the caseof DL-l-ch loropropa
| ,2-dioland the weak
ne-I ,2-diol,DL-3-bromopropanesubstrates,
the (m and ,/i,r valuesare reproducible
within 20?. 6valuesof I/.!, are relativeto ,/**{ry--r = 100asdeterminedby.eductionof NAD
ar pH 9.8 and 37 "C catalyzedby rn-Slycerol-3-phosphate
dchldrogenase-Absolurcvaluesof l/.,*,sly--l obtained(as describedin footnoted) were:
0.50OforC.mycodermo,0.203lotS.cereLisia?.0.823forE.coli.andO.49lforB.stearothermophilus.'IATP]=lmM.dThepredominan
of the aminegroupat pH 7.6 is indicated(p,(" = 9.4). "pH 9.5tThe triethanolaminebuffer (50 mM) wassubstitutedwith glycinebuffer (50 mM).
Assayconditionsare otherwiseas describedin footnotea.
OH
OH
cH2oH
0.044
0.049
8.0
5.5
5.0
100
t0'7,
84
66
30
0.041
0.042
0.047
5.7
8.7
2.9
t.l
100
a
J
phosphate(configurationundetermined). These two numbers
agree within experimentalerror and suggestthat the sample
containsonly one (*-ZVo) enantiomerof the organic phosphate.
Most of the organic phosphatesexaminedwere substratesfor
glycerol-3-phosphate
dehydrogenase
and were, as a result,assumed
to have the o configuration. In two cases(3-chloropropane-L,2diol-I -phosphateand 3-aminopropanewe
1,2-diol-3-phosphate),
establishedexplicitlythat the opposite(presumablyr-) enantiomer
was not acceptedby glycerol-3-phosphate
dehydrogenaseby'
demonstrating
that a racemicmixtureof l and o enantiomers
was
oxidizedonly to the extent of 50% by the enzyme. In one case
(o-butane-I ,2,4-triol-I -phosphate)the organicphosphatewas not
a substratefor glycerol-3-phosphate
dehydrogenase
and enantiomeric pure triols were preparedfrom >' and r--malicacid. Only
o-butane-l,z,4-triol derived from o-malic acid was phosphorylated
in the reaction catalyzeAby glycerol kinase. We have not explicitly
examinedthe absoluteconfigurationof phosphorylatedproducts
listed in Table I but not listed in Table IL We assume.however,
that all fall in the same stereochemicalseries.aT
Kinetics. The mechanismof phosphorylationof glycerolcatalyzedby glycerol kinasefrom rat liver is believedto follow an
ordered bi-bi mechanismin which glycerol binds first to the
enzyme,followed by ATP.Mg; sn-glycerol-3-phosphate
leaves
last.r2The patternof productinhibitionobservedin glycerolkinase
from f. coli suggestsa similar orderedmechanism.r2Although
straightforward in many details,the phosphorylationof glycerol
has one peculiarity: previousexaminationshave generatedbiphasic
double reciprocalplots,with the implicationof different values
of K* for ATP at low and high concentrationsof 41p.t2'2s A
relatedeffect has recentlybeenobservedwith glycerol itself with
glycerolkinasefrom adiposetissues.a8The origin of thesekinetic
peculiaritieshas not beenestablishedin detail, but they havebeen
suggested
to be due to two typesof sitesthat differ in their affinity
for ATP.r2 We have chosenconditionssuch that the ATP concentrationusedis higher than the highestreportedvalue of K..4e
Michaelis-Menten parameterswhich are not limited by cofactor
concentrationsare more relevant for use in enzyme-catalyzed
organicsynthesisthan thoseobtainedusingdilute reagents.Under
thesecircumstances,we observedgood linear behavior in Eadie-Hofstee plots (Figure 6). We have comparedthe kinetics
observedfor phosphorylationof glyceroland severalof its analoguesusingglycerolkinasefrom four different microbialsources.
We hoped in making this comparisonthat we might detect dif( 4 7 ) P r e l o g ,U . P u r e A p p l . C h e m . 1 9 6 4 , 9 , 1 1 9 - 3 0 .
(48) Barrera,L. A.; Ho. R.-J.Biochem.Biophys.Res.Commun.1979,86,
t45-52.
(49) The highestK,lor ATP is reportedfor glycerolkinasefrom E. c'oli.
T h e v a l u e sa r e i n t h e r a n s e0 . 1 - 4 m M . S e er e f 1 2 a n d 2 5 .
(D
HOH
o
HSXOH
\,/
\,/
v
n
Q
'=
ns,
o
8o
ao
o
c)
n
ao
-
a
6O
o
aa
t
o.o
o
o
a
m
a
I
roo
200
300
v/[s]
t.o
c)
o
+
tr
*r.o{o,
G)
tr
UN
o
o."o
(D
ol
oo
o
a
oo
o
a
roo
oo
200
v/[s]
300
plotsfor phosphorylation
of oL-3-mercaptoFigure6. Eadie-Hofstee
(lower)
propane-1,2-diol
(upper)and ol-J-methoxypropane-1,2-diol
of theglycerolkinaseusedin
catalyzed
by glycerolkinase.The sources
(O), S. c'ererisiae
(a), B.
werefrom C. ntye'oderma
theseexperiments
(O), and E. coli (tr).
stearothermophilus
ferencesin kinetic behavioramong theseenzymeswhich could
be used to advantagein organic syntheticprocedures.In fact,
the valuesobservedfor the different enzymeswere very similar
(Table III).
W e t h e r e f o r ec o n c l u d et h a t t h e a v a i l a b i l i t yo f
enzymesfrom differentmicrobialsourcesprovidesno advantage
in this particularsystem,and that the most attractiveenzymefor
most syntheticapplicationsis simply the least expensive,
Severalfeaturesof thesekinetic data dcservebrief comment.
First, Cleland and co-workershaveobservedfor glycerolkinase
from C. mycoderntathat K* for MgATP changedfrom 0.009 to
0 . 0 2 m M w h e n g l y c e r o lw a s r e p l a c e db y d i h y d r o x y a c e t o n e . 2 r ' s 0
We determinedK. tor ATP using glycerol kinase from E. coli
and pl-3-chloropropane-1,2-diol
as substrate(Kn,= 0.5 mM). The
similarity betweenthis valueand that observedfor glycerol(K.
(50) The K, for ATP for the followingsubstrates
are: l-glyceraldehyde.
0 . 0 4 m M ; o - g l y c e r a l d e h y d0e..0 0 7m M ; a n d o - p r o p a n e - 1 , 2 - d i 0
o .l ,1 m M .
J . A m . C h e m . S o c . ,V o l . 1 0 7 , . \ o . : 1 , 1 9 8 - \ 7 0 1 5
Glvcerol Kinase: Substrate Specificity
Y= OH
FI
I Z= H
HD
_/
YZ\
_\3H.
\.s
CO
'/ \
crr, oto;l
3=,$moll, ,
p r e f e r o b l yp o l o rg r o u p
(CHzOH
C ,H 2 C le, t c . )
X=O
NH
Figure7. Structuralcharacteristics
of substrates
for glycerolkinase.
= 0.3 mM) suggeststhat such effectsare not likely to be large
enoughto influenceour data.
The substratesincluded in Table III cover the range from the
most active observedin Table I to thosewith relatively low activity
(ol-butane-1,2,4-trioland ethyleneglycol). In going from highly
reactivesubstratesto inactive substrates,the valuesof K- increase
f r o m 0 . 0 4 t o 1 0 0 m M , w h i l e t h e v a l u e so f V ^ u * d e c r e a sbey a p proximatelya factor of 1000. The increasein K,n is not a matter
of great syntheticconcern;the enzyme is still active in highly
concentratedsolutionsof organic substrates,and the rates of
conversionare only greatly reducedat concentrationsconsiderably
lower than K.. The increase in K. can, however, become a
problem for the few racemic substrates(ot--propane-l .2-diol and
o t - - 3 - f l u o r o p r o p a n e - 1 , 2 - d iw
o lh) e
, r et h e e n a n t i o m e rt h a t i s n o t
a substrateinhibits the enzyme. The decreasein 2.,. is of more
importancefor syntheticuses;this decreasecorrespondsto a renl
decreasein the fastestpossiblerate of reactionand necessitates
largerquantitiesof enzymeto maintain usefulreactionrates. It
is, thus, ultimately the valuesof V^ * which limit the utility of
this enzyme as a catalyst for practical-scalesynthesiswith weak
substrates.
Conclusions
The data summarized in Table I permit the hypothesisof a
usefully detailed model summarizing the structural requirements
for acceptability as a substratewith glycerol kinase (Figure 7).
The position to which phosphatetransfer occurs can be either a
CH2OH group or a CH2NHT group, since it appearsthat the
enzyme is reluctant to accept groups other than hydrogen on the
carbon attached to the phosphorylatednucleophile. No secondary
hydroxyl group was phosphorylated. Compoundswith multiple
sites-if substrates-orient themselvessuch that the primary
hydroxyl group is phosphorylated. The group Y should clearly
be a hydroxyl group for highest activity. Substitution of hydroxyl
by fluorine or hydrogenleadsto poor substrates;substitutionby
NH2 or NHI* eliminatessub'strateactivity. The range of variation
accepted in the group Z is dependent on the enzyme source.
Hydrogen is obviously acceptable;the observation that dihydroxyacetonephosphatereacts (probably in the hydrated form)
suggeststhat OH is also acceptablefor enzyme from all four
sources.The activity of ol-2-methylpropane-1,2,3-triol
and 2(hydroxymethyl)butane1,2-diol(2-ethylpropane1,2,3-triol) with
glycerol kinase from C. mycoderma suggeststhat small alkyl
groupsare acceptablefor this enzyme,whereasevensmall alkyl
groups are too large for glycerol kinase from both E. coli and S.
cereuisiae. Other groups have not been systematicallyexamined.
The enzyme will accept a significant amount of variation in the
group R, although small, preferably polar groups lead to highest
rates. Variations are observedin substrateactivities for different
enzyme sour@s;the enzymesfrom S. cereuisiaeand C. mycoderma
acceptlarger substituentsthan the enzymesfrom E. coli and B.
st earothermophiI us.sl
The range of ratessummarizedin Table I is approximatelyl0a
from reactivity r = *** to reactivity r -- *. Compoundsshowing
reactivity r = *** provide the basisfor enzymatic transforma(5 I ) ot--3-Thiomethylpropane-1,2-diol
showedsubstrateactivitywith glycerol kinasefrom C. mycoderma,whereasno activity was observedfor glycerol
kinase from ^8.stearothermophilus.
tionssufficientlyefficientto generate100-gquantiticsof product;
thoseclassifiedas r -- ** can be phosphorylatedon l0-g scale
without major difficulty, and thoseclassifiedas reactivit\ r = *
are not practicalfor syntheticuses. Practicalillustrationsgiving
on the larger scalesare sumexperimentaldetailsfor procedures
paper.r' The examplesin this paper
marizedin the accompanying
are restrictedto small scales(<5-g), but the ratesgivenin Table
I correlatewith practicalsyntheticexperience.The group classified
r = * comprisessubstratesfrom three groups. In the first group
( e . g . ,o l - p r o p a n e 1
- , 2 - d i o al n d o l - 3 - f l u o r o p r o p a n e1-, 2 - d i o l ) n, o
substrateactivity is observedfor a oL mixture becauseweak
by inhibition due
activity of the oL enantiomerwas suppressed
t o t h e L e n a n t i o m e r .I n t h e s e c o n dg r o u p ( e . g . ,a c e t o la n d o l glycericacid), the substrateinducessignificantATPaseactivity
( > 4 0 % o f t o t a l a c t i v i t y ) i n g l y c e r o lk i n a s ec o n c u r r e n t l yw i t h
substrate phosphorylation. In the third group (e.9., DL-l/1,2-dioland glycidol),the substrateand
acetyl-3-aminopropaneits hydrolysisproductgeneratea mixture in which phosphorylation
catalyzed by glycerol kinases produces nonspecific mixtures.
Practical examplesof synthesesare summarizedin an accompanying paper.rr The group classifiedr = 0 includesthe crude
preparationsof compoundsdescribedin the Experimental Section
which contain impurities. It is, of course,possiblethat small
quantitiesof adventitiousimpurities in the crude preparations
t e s t e df o r s u b s t r a t ea c t i v i t y a c t a s i n h i b i t o r s . l n t h e c a s e o f
o t - - , N - m e t h y l p r o p a n e - 1 . 2 -b
da
i oslc. do n t h e r H N M R s p e c t r u m ,
w e e s t i m a t et h c a n r o u n o
t l ' i n r p u r i t lt o b e l e s st h a n l 0 ' 7 : f b r o t h e r
c o m p o u n dlso c l : o f i n r p u r i t i c(su n l c : so t h c r rirs ci n d i c a t c)dw e r e
l o uer .
. ' \ n i n t c r e s t r n rgc : u l l t h a t c n r c r g c :I - r o r nt h r : * o r k i s t h e o b servation
t h a t h r g h l r : c l c c t rcr p h t r : p h t r lrar t i o no n n i t r o g e nc a n
b e a c h i e r e di n o t - - l - e n r i n o p r o p aln. lc--d r o l . T h i . : u b s t r a t ea c t i v i t y
w a s n o t d u p l i c a t e db 1 o t h e r s t r u c t u r a l l la n a l o g o u as m i n e sa l l r o p a n c - l - oiln d u c e d. . \ T P a s e
act h o u g ho t - - 3 - a m i n oI -- m e t h o x p
t i v i t y . s 2 T h e i n t e r e s ti n t h i s o b s e r v a t i o ni s l e s si n t h c u t i l i t y o f
t h e r e a c t i o ni t s e l f t h a n i n t h e i m p l i c a t i o nt h a t a n e n z \ m e m a y
have useful activity under nonphysiological
conditions*'hich is
masked under physiologicalconditionsby protonation of nucleophiliccenters.
ln summary, this study bearson two concerns. First, it establishesthat the range of structureswhich are in substratesfor
glycerol kinase is wider than has been suggestedin previouswork.
In particular,the substrateactivity of or--3-chloropropane1,2-diol
This
is relevantto the antifertility activity of this substance.34'35
work alsosuggeststhat glycerolkinaseshouldbe a usefulcatalyst
in syntheticorganicchemistry. It is possiblein one stepto prepare
chiral analoguesof glycerol from
a number of phosphorylated,
the racemicmixtures. Thesematerialsshouldfind usein synthesis
of phosphorylatedorganic substances(especiallyanaloguesof
phospholipidsrs).
Experimental Section
Materials.Glycerolkinasefrom E. coli wasobtainedas lyophilized
powderfrom Sigma. Glycerolkinasefrom C. mycoderma
wasobtained
suspension
in ammoniumsulfatefromSigmaandfrom
asa crystalline
wereobserved
Boehringer
Mannheim.No differences
in the kinetic
parametersfor the C. mycodermaenzymefrom thesetwo sources.
wasobtainedasa solution
Glycerolkinasefrom B. stearothermophilus
in Tris buffer from BoehringerMannheim. Glycerolkinasefrom S.
wasobtainedfrom Genzyme.Otherenzymes
cereuisiae
andbiochemicals
wereobtainedfrom Sigma.All enzymes
wereusedasreceived;
absolute
puritieswerenot established.
werereagentgradeand used
Chemicals
withoutfurtherpurification
unlessotherwise
indicated.ol-3-Chloropropaneweredistilledbefore
1,2-diol
and ol-3-bromopropane1.2-diol
(distillation
wasnot necessary
usefor kineticmeasurements
for the llP
N M R a s s a y s a) ;q u e o usso l u t i o nosf o r - 3 - c h l o r o p r o p a n e - 1 , 2a-nddi o l
( 5 2 ) T h e a c t i v i t yo f o - 6 - a m i n o - 6 - d e o x y g l u cionspeh o s p h o r y l a t i ocna t a was examinedby a modificationof the assaydescribed
lyzed by hexokinase
in the ExperimentalSection;glycerolkinasewassubstitutedwith hexokinase
from yeast. After 24 h in a signalat 9 ppm was observedin the rrP NMR
spectrum. The reactivity was less than 20 times the ATPase activity of
hexokinasefrom yeast(as determinedby integrationof the 3rPNMR peak
heightsof the phosphoramidate
and the inorganicphosphateproducedin the
r e a c t i o n ) .N o i s o l a t i o n w
s e r ec a r r i e do u t .
7016
J. Am. Chem. Soc., Vol. 107, No. 24, 1985
o L - 3 - b r o m o p r o p a n e - 1 , 2 - d iw
o le r e f r e s h l y p r e p a r e db e f o r e u s e . W a t e r
w a s d i s t i l l e d t w i c e . t h e s e c o n dt i m e f r o m g l a s s .
Methods. Spectrophotometricmeasurementswere performed at 25
" C u s i n g a P e r k i n - E l m e r M o d e l 5 5 2 s p e c t r o p h o t o m e t eerq u i p p e dw i t h
y as roua c o n s t a n t - t e m p e r a t u rcee l l . P h o s p h o r u sN M R s p e c t r o s c o p w
t i n e l y d o n e o n a 4 0 . 4 8 - M H t ( 2 3 - T ) V a r i a n i n s t r u m e n ti n l 2 - m m N M R
t u b e s . S a m p l e sc o n t a i n e d2 0 V ad e u t e r i u mo x i d e a s i n t e r n a l N M R l o c k .
compoundswere reported as
Chemical shifts for phosphorus-containing
positiveupfield relativeto external 85% phosphoricacid; the temperature
was 30 oC unlessnoted otherwise. Accumulation parametersused for
a s s a y sw e r e : p u l s ea n g l e .2 l o ; p u l s ed e l a y , 1 . 5s ; a c c u m u l a t i o nt i m e , 1 . 0 2
s ere carriedout usinga 90o
s . Q u a n t i t a t i v er l P N M R m e a s u r e m e n t w
p u l s ea n g l ea n d a 2 - m i n p u l s ed e l a y ,a n d a n i n t e r n a l s t a n d a r do f k n o w n
y as
c o n c e n t r a t i o n( s o d i u mp h o s p h a t e ) .C a r b o n - 1 3N M R s p e c t r o s c o pw
routinelydone on a 67.8-MHz (63 T) Jeol spectrometerin a 5-mm NMR
tube. Samples( 100-200 mg/mL) were dissolvedin deuterium oxide and
rrC chemical shifts were reported relative to external sodium 2,z-dim e t h y l - 2 - s i l a p e n t a n e - 5 - s u l f o n a( D
t eS S ) . T h e s p e c t r aw e r e c o m p l e t e l y
proton decoupledand the accumulationparametersused for the spectra
w e r e : p u l s ea n g l e , 9 0 o ;p u l s ed e l a y , 1 . 4 5s ; a c c u m u l a t i o nt i m e , 0 . 5 5 s .
Mass spectra were recorded on an AEI MS9 mass spectrometer at
50-100'c.
I'P NMR Assay for SubstrateActivity with Glycerol Kinase. The 'rP
N M R a s s a yc a r r i e do u t i n a l 2 - m m N M R t u b e c o u l d c o n r c n i e n t l r
o b s e r v eI t o l 0 i . t n r ool f p h o s p h o r u s - c o n t a i n icnogm p o u n d .T h c b u f i e r
a s s a l a t p l { l . - i u a s p r c p a r e da s f o l l o i r s . T r i lor the riP \\{R
c h l o r i d et 0 l 5
e t h a n o l a m i nhey d r o c h l o r i d1e- s . 5g7. - j 0m m o l ) .m a g n e s i u m
g , 1 . 2 - sm m o l ) , P E P t r i s o d i u ms a l t ( 1 . 3 ag . 6 . 5 m m o l ) . a n d . ' \ T P t r i in uater (100mL) and
s o d i u ms a l t ( 0 . 3 9g , 0 . 6 5 m m o l ) * e r e d i s s o l v e d
t h e p H w a s a d j u s t e dt o 7 . 5 w i t h 2 M N a O H . T h e r r P N M R a s s a l
c a r r i e d o u t a t h i g h p H ( w h e n s u b s t r a t e sc o n t a i n a m i n e g r o u p s ) u s e da
modified buffer in which the triethanolamine hydrochloride was replaced
b y g l y c i n e ( 2 . 2 5 g , 3 0 m m o l ) a n d t h e p H w a s a d j u s t e dt o 9 . 5 .
1,2-diol followed this
A representativeassayfor oL-3-mercaptopropanecourse. Triethanolaminebuffer (0.3 M, 3.2 mL) containing magnesium
c h l o r i d e( 1 2 . 5 m M ) , P E P ( 6 5 m M ) , a n d A T P ( 6 . 5 m M ) w a s a d d e dt o
d e u t e r i u mo x i d e ( 0 . 7 m L ) . o r - - 3 - M e r c a p t o p r o p a n e - 1 , 2 - d( 0i o.Il m L , 0 . 8
mmol) was added to the buffer solution and generateda substrateconc e n t r a t i o no f 0 . 2 5 M . T h e a s s a yw a s s t a r t e d b y a d d i t i o n o f p y r u v a t e
k i n a s e( l U ) a n d g l y c e r o l k i n a s e ( 5 U ) , a n d w a s l e f t f o r 2 4 h a t r o o m
temperature. The 3rPNMR chemicalshifts of the assaysolutionwithout
s u b s t r a t ea r e : A T P t - 5 ( d ) , - 1 0 ( d ) , - 1 9 ( t ) 1 , P E P [ - 1 ( s ) ] . A f t e r 2 4
trP NMR spcctrum
h t h e f o l l o w i n gc h e m i c a ls h i f t sw e r e o b s e r v e di n t h e
of the ol-3-mercaptopropane-1.2-a
d si osla rs o l u t i o n :A D P [ - 5 ( d ) . - 1 0
( d ) ] , t h e o r g a n i c p h o s p h a t ep r o d u c e db 1 p h o s p h o r l l a t i o no i o r - 3 m e r c a p t o p r o p a n e - l . l - d i o[ l- ] 5 ( t . ' ' l - . * , = 6 . - 1H z ) 1 . F o r r e f e r e n c et.h c
3 r Pc h e m i c a ls h i f t o i i n o r g a n i cp h o s p h a t cu a s l . - i p p n r .
P u r i t y . T h e e n a n t i o m e r ip
c u r i t r o i t h e i s o l a t e dn i a t c r i a l si r a s d e t e r m i n e d b y q u a n t i t a t i v ee n z y m a t i ca n a l y s i s .T h e c h e m i c a lp u r i t l o f t h e
o r g a n i cp h o s p h a t e *sc r e d e t e r m i n e db y q u a n t i t a t i v er r P \ \ { R . T h e
c h a r a c t e r i z a t i o nu
sere carriedout as follows.
E n z y m a t i cA n a l y s i s . T h e p r o c e d u r ef o r q u a n t i t a t i v ed e t e r m i n a t i o n s
o f . s n - g l y ' c e r o l - 3 - p h o s p hw
a taes b a s e do n t h e g l y c e r o l - 3 - p h o s p h a tdee Since most of the snh y d r o g e n a s cc a t a l y z e d r e d u c t i o n o f N A D .
g l y c e r o l - 3 - p h o s p h a taen a l o g u e sa r e s u b s t r a t e sf o r g l y c e r o l - 3 - p h o s p h a t e
t ea s
d e h y d r o g e n a s et h, e p r o c e d u r ef o r a s s a yo f s n - g l y c e r o l - 3 - p h o s p h aw
e x p a n d e dt o i n c l u d e t h e s e a n a l o g u e s . T h e a s s a y w a s c a r r i e d o u t a s
assay
A representative
with minor modifications.43'45
describedpreviously'
for o-3-aminopropan1
e ,- 2 - d i o l - 3 - p h o s p h af toel l o w s . A b u f f e r c o n t a i n i n g
h y d r a z i n ea n d g l y c i n e ( 2 . 0 0 m L , 0 . 0 4 M h y d r a z i n e ,0 . 5 M g l y c i n e ,p H
9 . 8 ) a n d N A D ( 0 . 1 m L , 3 l m M ) w a s p l a c e di n a 3 - m L c u v e t t e . T h e
c u v e t t e w a s e q u i l i b r a t e df o r 2 - 4 m i n a t 2 5 o C , g l y c e r o l - 3 - p h o s p h a t e
at 340nm
d e h y d r o g e n a s(e2 0 p L , 2 0 U ) w a s a d d e d ,a n d t h e a b s o r b a n c e
was recorded(exnoH= 6220 M-lcm l). When a steadyabsorbancelevel
( b a s e l i n e )h a d b e e n r e a c h e d ,a s m a l l v o l u m e ( 0 . 1 m L ) o f t h e s a m p l e
s o l u t i o nw a s a d d e dt o t h e a s s a ys o l u t i o na n d t h e i n c r e a s ei n a b s o r b a n c e
w a s r e c o r d e d . A i t e r 5 t o l 0 m i n n o f u r t h e r i n c r e a s ei n t h e a b s o r b a n c e
w a s o b s e r v e d . T h e s a m p l e s o l u t i o n c o n t a i n e do - 3 - a m i n o p r o p a n e - 1 , 2 d i o l - 3 - p h o s p h a tbea r i u m s a l t ( 3 2 m g ) d i s s o l v e di n w a t e r ( 2 5 m L ) . T h e
a s s a ) sl \ e r e r u n i n d u p l i c a t e s( o r t r i p l i c a t e si f m o r e t h a n 3 % v a r i a t i o n
w a s o b s e r v e di n t h e f i r s t t w o d e t e r m i n a t i o n s ) .T h e a b s o r b a n c ea t 3 4 0
n n r w a s m c a s u r e da g a i n s ta b l a n k , a l t h o u g ht h i s c o n t r o la p p e a r e dn o t t o
b e c r i t i c a l f o r t h e d e t e r m i n a t i o n s .I n t h e b l a n k , w a t e r w a s s u b s t i t u t e d
f o r t h e s a m p l es o l u t i o n .
r r P N M R . T h e p h o s p h o r u s - c o n t a i n i nc g
o m p o u n d sw e r e
Quantitatire
a s s a y e dq u a n t i t a t i v e l yb' y i n t e g r a t i o no f t h e i r l r P N M R s i g n a l sa n d
c o m p a r i s o no f t h c o b s e r v e di n t e n s i t i e sw i t h t h a t o f a n i n t e r n a ls t a n d a r d
o f k n o w n c o n c e n t r a t i o n . 5 lT h e 3 l P N M R s D e c t r aw e r e r e c o r d e di n 2 0 %
( 5 3 ) B a r a n y .M . ; G l o n e k ,T . M e t h o d sE n z y m o l .1 9 8 2 ,8 J , 6 2 4 - 1 6 .
Crans and Whitesides
d e u t e r i u mo x i d e ( p H 7 - 9 ) u s i n g a p u l s ea n g l e o f 9 0 o a n d a p u l s ed e l a y
of 2 min.5a The data were processedtwice and the averagewas reported.
A representativesample solution was prepared as follows: n-3-thiomethylpropane-I ,2-diol-I -phosphatebarium salt ( I 24 mg) was dissolved
i n w a t e r ( 3 m L ) b y a d d i t i o n o f e x c e s ss o d i u m s u l f a t e ( 1 5 0 m g ) . T h e
precipitated barium sulfate was removed by centrifugation, and the
p r e c i p i t a t ew a s w a s h e dw i t h d e u t e r i u mo x i d e ( 0 . 8 m L ) . A f t e r t h e p r e cipitate was removedfrom the deuterium oxide, the supernatantand the
3rP
deuteriumoxide wash were placedin a l2-mm NMR tube and a
(without
processed
were
twice
NMR spectrum was recorded. The data
sensitivity enhancement) and the average was reported. An internal
s t a n d a r d ,i n o r g a n i c p h o s p h a t e ,w a s a d d e d ( i n t h e c a s e o f o l - 3 - t h i o e t h y l p r o p a n e - 1 , 2 - d i o l - l - p h o s p h a t e ,m
0 .L5 o f 0 . 4 M m o n o s o d i u mp h o s phate solution) and another rrP NMR spectrumwas recorded. The
i n t e n s i t i e si n t h e 3 r P N M R s p e c t r u mw e r e m e a s u r e da n d t h e p u r i t y o f
t h e o r g a n i cp h o s p h a t ew a s c a l c u l a t e db a s e do n t h e i n o r g a n i cp h o s p h a t e
u s e d a s i n t e r n a l s t a n d a r d . T h e i n o r g a n i c p h o s p h a t eu s e d a s i n t e r n a l
s t a n d a r d h a d p r e v i o u s l yb e e n c a l i b r a t e d a g a i n s t o r - - g l y c e r o l - 3 - p h o s p h a t e . 5 5 T h e s e n s i t i v i t yo' f t h e m e t h o d w a s e x a m i n e db y a d d i n g i n creasingamountsof inorganicphosphateto a samplesolutioncontaining
disodiumsalt. This assaywas
knoun amountsof ot--glycerol-3-phosphate
able to detect inorganic phosphateat a concentrationequal to 2 mol Vo
Impurities
of the major phosphatecomponent(ot--glycerol-3-phosphate).
* e r e t h c r e f o r ed e t e c t e da t t h e l e v e lo f 2 m o l % b u t t h e o v e r a l la c c u r a c y
o l t h e q u a n t i t a t i v ed e t e r m i n a t i o n sw a s n o t h i g h e r t h a n 5 V o .
Kinetic f)eterminations. Initial rates of enzymatic reactionswere all
at 25 "C. The phosphorylgroup transfer
deternrrnedbv UV spectroscopy
f r o m A T P t o s u b s t r a t ew a s c o u p l e dt o t h e o x i d a t i o n o f N A D H u s i n g
p h o s p h o e n o l p l r u v a t ep,y r u v a t ek i n a s e( 8 . C . 2 . 1 . 1 . 4 0 ) ,a n d t - - l a c t i cd e h y ' d r o g e n a s(eE . C . 1 . 1 . 1 . 2 7 ) .T h e f o r m a t i o no f N A D ( o r t h e d i s a p p e a r a n c eo f N A D H ) w a s m o n i t o r e d a t 3 4 0 n m . S o l u t i o n sc o n t a i n e d
triethanolaminehydrochloride(50 mM, pH 7.6), ATP disodiumsalt (5.0
m M ) , N A D H ( 0 . 1 6 m M ) , P E P t r i s o d i u ms a l t ( 3 . 0 4 m M ) , m a g n e s i u m
s u l f a t e( 1 5 . 6 m M ) , p o t a s s i u mc h l o r i d e( 1 . 0 4 m M ) , p y r u v a t ek i n a s e( 2 0
U ) , t - - l a c t i cd e h y d r o g e n a s (e4 0 U ) , a n d 0 . 0 1 - l U o f g l y c e r o lk i n a s e ' 2 i
T h e t o t a l a s s a yv o l u m e w a s 2 . 5 m L . T h e m e a s u r e m e n t so f r a t e s o f
reaction for glycerol analogueswere done at the following substrate
c o n c e n t r a t i o n s0: . 5 , 1 , 2 , 3 , 5 , 1 0 , 1 5 , a n d 2 0 m M . I n t w o c a s e s( p r o p a n e - 1 , 3 - d i oal n d g l y c e r o l ) .t h e K . v a l u e sw e r e s i g n i f i c a n t l yd i f f e r e n t
from thoseof the other glycerolanalogues.The substrateconcentrations
w e r e v a r i e d f r o m l 0 t o 5 0 0 m M f o r p r o p a n e - 1 , 3 - d i oal n d f r o m 0 . 0 1 t o
0.2 mM for glvcerol. ATP and the magnesiumion were presentin excess
a t a l l t i m e s . R e a c t i o n sw e r e s t a r t e db y a d d i t i o n o f s u b s t r a t e . C o n t r o l
c r p e r i r n c n t su e r c c a r r i e do u t t o e n s u r et h a t t h e r a t e - l i m i t i n gs t e pw a s
t h a t c a t a l l z e d b r g l r c e r o l k i n a s e . T h e g l y c e r o la n a l o g u e sh a d n o e f f e c t
o n t h c r a t e so t ' r e a c t i o no f p y r u v a t ek i n a s ea n d t . - l a c t i cd e h y d r o g e n a s e ;
r r t c s ( r f r c a c t i o nd i d n o t c h a n g eb y t h e a d d i t i o no f g l y c e r o la n a l o g u e si n
. r \ \ . r \ s i n u h i c h g l y c e r o la n d g l y c e r o lk i n a s eh a d b e e n s u b s t i t u t e dw i t h
g l u c o s ea n d h e x o k i n a s e .A s t a n d a r da s s a yf o r g l y c e r o lk i n a s ea c t i v i t y
using 0..1m\{ gl1'cerolas substratewas performedbefore and after each
s c r i e so f c r p er i m e n t s . O n e u n i t o f g l y c e r o lk i n a s ea c t i v i t y i s d e f i n e da s
t h e a n r o u n to f en z y ' m et h a t i s r e q u i r e dt o c a t a l y z et h e t r a n s f o r m a t i o no f
I p m o l o l g l v c e r o lp e r m i n t o s n - g l y c e r o l - 3 - p h o s p h aat tep H 9 . 8 a n d 3 7
oC. The commerciallyavailableglycerolkinasefrom E. coli was assayed
accordingto the aboveprocedure.56Assay of glycerolkinasefrom other
sourceslollowed this standard. The assayconcentrationswere as follows:
h y d r a z i n e( 0 . 8 8 M ) , g l y c i n e( 0 . 1 8 M ) , m a g n e s i u mc h l o r i d e( 0 . 2 5m M ) ,
A T P ( 1 . 4 m M ) , N A D ( 0 . 2 5 m M ) , g l y c e r o l( 1 . 5 m M ) , a n d g l y c e r o l - 3 p h o s p h a t ed e h y d r o g e n a s (e1 0 U ) .
Glycerol Kinase Catalyzed Phosphorylationof Glycerol Analogues:
General. The synthesesof phosphorylatedglycerol analogueswere carried out on scalesfrom 0.2 to I mmol using in situ ATP regencrationand
solubleenzymes. PEP was usedas the ultimate phosphorylgroup donor.
The glycerol kinaseused for theseenzyme-catalyzedsyntheseswas from
S. cerecisiaeor from E. coli. The syntheseswere performed under similar
n a l o g u e s .T h r e c r e p r e c o n d i t i o n sf o r a l l t h e s n - g l y c e r o l - 3 - p h o s p h aat e
s e n t a t i v e s y n t h e s e s( n - 3 - c h l o r o p r o p a n Ie,-2 - d i o l -I - p h o s p h a t e ,o - 3 a m i n o p r o p a n e1- , 2 - d i o l1- - p h o s p h a t ea, n d e t h y l e n eg l y c o l p h o s p h a t e )i l l u s t r a t et h e v a r i a t i o n si n t h e r e a c t i o n s .T a b l e I V s u m m a r i z e st h e d a t a
f o r o t h e r s n - g l y c e r o l - 3 - p h o s p h aat en a l o g u e s .
nl-3-Chloropropane-I .2-diol
o-3-Chloropropane-1,2-diol-l-phosphate.
( r e a g e n gt r a d ef r o m A l d r i c h , 0 . l 0 m L , 1 . 2m m o l ) , P E P m o n o p o t a s s i u m
s a l t ( 8 3 m g , 0 . 4 0 m m o l ) , a n d A T P d i s o d i u ms a l t ( 2 5 m g , 0 . 0 4 0m m o l )
w e r e d i s s o l v e di n a 0 . 1 M s o l u t i o no f t r i e t h a n o l a m i n e( 3 m L ) c o n t a i n i n g
m a g n e s i u mc h l o r i d e( 1 0 m M ) a n d d e u t e r i u mo x i d c ( 0 . 7 m L ) . T h e p H
( 5 4 ) G l o n e k .T . ; V a n W a z e r ,J . R . " / . P h y s . C h e m . 1 9 7 6 . 8 0 , 6 3 9 - 4 3 .
( 5 5 ) G l o n e k . T .J . A m . C h e m .S o c .1 9 7 6 ,9 8 , 1 0 9 0 - 2 .
( 5 6 ) G l y c e r okl i n a s eS, i g m a ,P r o d u c N
t o . G - 4 5 0 9 ,L o t N o . l 0 l F - 6 8 0 5 .
d n a p r o d u c tc h a r t .
A s s a ya n d p r o d u c ta r e d e s c r i b e o
GlycerolKinase: SubstruteSpecificity
J. Am. Chem.Soc.,Vol. 107,No. 24, 1985 7Ol1
Trbl€IV. Summary
of Isolated
YieldsandExperimental
Conditions
for Phosphorylation
Catalr'zed
b\ (;lycerolKinase'
product
-2OoP
OH
scale
(mmol)
cH20H
H
cH2cl
CH2Br
ch2sH
cH2ocH3
cH2scH3
cH2ocH2cH3
cH2cH3
cH2cH2oH
CH2CN
cH(cHr)oH
RTD
(days)
GK'/PK
(U/U)
I
specia
conditions
rrC NMR datad
'/r*..
'/r*.
yield' (%)
0.22
I
20110
7.3
95
0.47
7.3
6
1s0l2o
Pi,
60
1.3
0.42
2
20lt0
96
0.53
2
95
201t0
6.1
11
0.61
2
t.-)
12
I 00/20
Pi'
0.25
2
25120 Pi'
t.J
IJ
0 . 35
l 00i 20
92
0.23
251t0
88
0.22
1 5 0 1 2 0 P . ' ,. S . r ' t ' r e ?
t.)
94
0.48
200/r 00
9.2
96
0.10
100120 P,'
15
0.20
l soi r 00
[,. t'rtlis
37
-5.5
87
ro.PN
0.29
100/30 pt{ 10.0/'
1.6
7.8
81
'The reactionwascarriedout asdescribedfor DL-3-chloropropaneI ,2-diolunlessotherwiseindicaredlspecialconditions).'The indicatedreaction
times(RT) are upperlimits fo. completionof the reactions."The reactionswerecarriedout wilh glycerolkinasefrom S. rer€r,rideunlessothe.wise
indicaled. Similar resultsare obtainedwith glycerolkinasefrom E. ro1i. r'The productstructureindicaredwasverifiedby observingthe two- and
three-bondcouplingconstanlsofth€cartrcnsaandptophosphorusintherrcsp€ctrum.'Theisolatedlieldsarecalculatedbasedonthe
phosphorylgroup donorsaddedin the reaction- /During the reactioninorganicphosphateformed. The inorganicphosphat€was .emovedby
precipitationas barium phosphatebeforethe organicphosphatewas isolated(as describedfor ethyle e glycol phosphate).ronly the indicated
ghcerol kinasecatalyzedthe formationof product. tThe reactionwascarriedout at pH 10.0.
A
t
A
was adjustedto 7.5 and the reaction was started by addition of glycerol
kinase(20 U) and pyruvatekinase(10 U). After 3 days the reactionwas
c o m p l e t e ;P E P a n d A T P w e r e n o l o n g e r o b s e r v e di n t h e 3 r P N M R
s p e c t r u m . T h e s o l u t i o nw a s p a s s e dt h r o u g h c h a r c o a l( 0 I g ) t o r e m o v e
A D P a n d e n z y m e s .B a r i u m c h l o r i d e( 0 . 2 a g , 1 . 0 m m o l ) a n d e t h a n o l1 2 - 5
m L , 9 5 V o )w e r e a d d e d t o t h e s o l u t i o n t o p r e c i p i t a t e r h e o - - l - c h l o r o p r o p a n e - 1 , 2 - d i o l - l - p h o s p h abtaer i u m s a l t . A f t e r t h e s o l i d u a s r e m o v e d
b y c e n t r i f u g a t i o na n d d e c a n t i n gt h e s u p e r n a t a n t t. h e s o l i d u a s * a : h e d
o n c e w i t h e t h a n o l ( 2 5 m L , 9 5 V o )a n d d r i e d o v e r C a S O o a r I r o r r o \ c r n i g h t . T h e w h i t e s o l i d w a s 9 7 7 op u r e a s d e t e r m i n e db y e n z rm a r i c a s s a \
and contained more than 95Va organic phosphate as determined b1
q u a n t i t a t i v er r P N M R ( n o o t h e r p h o s p h a t e - c o n t a i n i ncgo m p o u n du ' a s
observed). The yield basedon combined phosphorylgroup donors was
9 6 V o :t H N M R ( D z O , p H - l ) 6 ( D S S ) 3 . 9 5 - 4 . 1 5( m , I H ) , 3 . 6 0 - 3 . 8 , s
( m , 4 H ) ; r 3 CN M R ( D 2 O ,p H - l ) 6 ( D S S ) 7 5 . 3( d , 3 J = 7 . 3 H 2 ) , i 0 . 4
( d , 2 " r= 3 . 7 H z ) , 4 9 . 9 ( s ) ; I ' p N M R ( D z O , p H g . 2 ) 6 4 . 2 ( t , 3 J = 6 . 5
Hz).
o-3-Aminopropane-1,2-diol-3-phosphate.
ol-3-Aminopropane- 1,2-diol
( 0 . 1 0 m L , 1 . 3m m o l ) , P E P m o n o p o t a s s i u m
salt (80 mg,0.39 mmol),and
ATP disodium salt (25 mg, 0.040 mmol) were dissolvedin 0.1 M glycine
( 4 m L ) c o n t a i n i n gm a g n e s i u mc h l o r i d e ( 1 0 m M ) a n d d e u t e r i u mo x i d e
(0.7 mL). The pH was adjustedto 10.3 and the reaction was started by
a d d i t i o n o f g l y c e r o l k i n a s e ( 1 0 0 U ) a n d p y r u v a t e k i n a s e( 3 0 U ) . T h e
reaction was complete after 2 days; the lrP NMR spectrum showed no
remaining PEP or ATP. The solution was passedthrough charcoal (0.1
g ) t o r e m o v eA D P a n d e n z y m e s . B a r i u m c h l o r i d e ( 0 . 1 2 g , 0 . 5 0 m m o l )
a n d e t h a n o l ( 2 5 m L , 9 5 7 o ) w e r e a d d e d t o p r e c i p i t a t et h e o r g a n i c p h o s phate. The suspensionwas stirred at 0 "C for 30 min and the solid was
separatedby centrifugation. The supernatantwas discardedand the solid
was washed once with ice-cold ethanol (50 mL, 95Vo). The suspended
precipitatewas isolatedby centrifugationand the supernatantdiscarded.
The solid was dried over CaSOa at I torr overnight. The resulting barium
o - 3 - a m i n o p r o p a n e - 1 , 2 - d i o l - l - p h o s p h a( 2t e0 m g , 0 . 3 8 m m o l ) w a s 9 8 V o
pure as determined by enzymatic assayand contained more than 95%
organic phosphateas determined by quantitative 3rP NMR (no other
phosphate-containingcompound was observed). The yield based on
c o m b i n e dp h o s p h o r y lg r o u p d o n o r s w a s 8 7 V o : t H N M R ( D z O , p H I l )
6 ( D S S ) 3 . 7 0 - 3 . 8 0( m , I H ) , 3 . 5 0 - 3 . 6 5( m , 2 H ) , 2 . 7 5 - 2 . 9 0( m , 2 H ) ;
r r C N M R ( D r O , p H l l ) 6 ( D S S ) 7 6 . 8( d , 3 J = i . 3 H z ) , 6 8 . 1 ( s ) , a 9 . 0
I P N M R ( D z O ,p H l 1 ) 6 9 . 4 ( t , 3 J = 8 . 9 H z ) .
@ , 2 J = 1 . 6H z ) ;
Ethylene Glycol Phosphate. Ethylene glycol (0.25 mL, 0.45 mmol),
PEP monopotassium
salt (150 mg,0.70 mmol), and ATP (60 mg,0.090
mmol) were dissolved in 0.1 M triethanolamine (6 mL) containing
magnesiumchloride (10 mM) and deuteriumoxide (l mL). The pH was
adjustedto 7.8 and solubleglycerol kinase (150 U) and pyruvate kinase
(20 U) were added to the solution. After 2 days excessethyleneglycol
(0.25 mL, 0.45 mmol) was added to the reaction mixture, and after 4
more days the reaction was complete; 3lP NMR spectrum showed no
remaining ATP or PEP. The solution was passedthrough charcoal (0.1
g ) t o r e m o v eA D P a n d e n z y m e s . B a r i u m c h l o r i d e ( 2 4 m g , 0 . l 0 m m o l )
was added to the reaction mixture and most of the inorganic phosphate
precipitated as barium phosphate. The precipitate was removed by
centrifugation, and then barium chloride (240 mg, 1.0 mmol) and 5
volumes of ice-cold acetone were added to the supernatant. The pre-
c i p i t a t e w a s a l l o w e d t o s e t t l ef o r 3 0 m i n a t 0 o C a n d c e n t r i f u g e d . T h e
supernatantwas decantedand the solid was washedonce with acetone
( 2 0 m L ) . T h e r e s u l t i n gb a r i u m e t h y l e n eg l y c o lp h o s p h a t e( 1 6 0 m g ) w a s
d r i e d o v e r C a S O . a t I t o r r o v e r n i g h t . A s d e t e r m i n e db y q u a n t i t a t i v er r P
\ M R . t h e p u r i t r u a s 8 l ' 7 a n d c o r r e s p o n d etdo a . , ' i e l dt - t 6
f 0 ( 7 b, a s e do n
c o n r b i n c dp h o s p h t r ir ru r o u pd o n t l r s T h c n t r r l o ri m p u r i t ; . a s d c t e r m i n e d
t
'
P
:
t
l
\ \ l R . \ \ i r \ i n , ) r s i l n ip. h ( r \ p h i t t( cl i ' l ) :
t f . { R ( D . O .p H 7 . 5 )
b)
-a I
-i
\ ) r n i .I t l i . I 6 : I
o t l ) S S )- 1
r n r .I F l ) :r ' C \ M R ( D 2 0 ,p H
- . 5 ) ; ( D S S ) 6 -Q
r : . / r , , =, .. r H z ) . 6 J - ( t / P o , , ,= 1 . 3 H 2 ) : 3 l P N M R
' . / 1 , , , =,
r D - O .p l l i t ' J I r t
ri i r Ilzt
o r - 3 - ( ' h l o r o p r o p a n e - 1 . 2 - d i o l - l - p h o s p h\ \a' rt lesp r e p a r e df r o m e p i c h l o r o h r d r i na n d p h o ' p h o r i e. r c r da c c o r d i n pt o t h e p r o c e d u r eo f Z e t z s c h e
a n d A e s c h l i m a n n :r' '-C \ \ 1 R ( D : O . p t i - t r ; t D S S ) 7 4 . 2( d , 3 J =
'iP
7 . , 1H z ) . 7 0 . 6( d . : J = 3 . 1H z ) . . 1 9 ( . ) :
\ M R ( D : O . p H 9 . 8 )6 4 . 4
(t. r.r = 6.8 Hz).
or--3-Aminopropane1,2-diol-3-phosphate.Phosphorusoxychloride ( I .9
m L , 2 0 m m o l ) a n d o l - 3 - a m i n o p r o p a n e - 1 , 2 - d i o( 1l . 9 g , 2 0 m m o l ) w e r e
dissolvedin acetonitrile (25 mL) and cooled in a salt water-ice bath.
Triethylamine (2.0 g, 20 mmol) was added dropwiseto the solutionover
a period of 30 min. After being stirred for t h at 0 oC the solution was
s l o w l y a d d e d t o a n a q u e o u ss o l u t i o no f N a O H ( 2 5 m L , 2 M ) a t 0 ' C .
The reactionmixture was stirred for 30 min at 0 oC and for an additional
2 h at ambient temperature. Barium chloride (2.4 g, l0 mmol) was
a d d e d t o p r e c i p i t a t et h e i n o r g a n i cp h o s p h a t e .T h e p r e c i p i t a t ew a s r e m o v e db y f i l t r a t i o n . A d d i t i o n a lb a r i u m c h l o r i d e( 2 . 4 e , l 0 m m o l ) a n d
ethanol(75 ml-. 957.) were addedto the filtrate to precipitatethe barium
n l - 3 - a m i n o p r o p a n eI-. 2 - d i o l - 3 - p h o s p h a t eT. h e s u s p e n s i o w
n as allowed
t o s e t t l ef o r 3 h a t 0 o C b e f o r et h e s o l i d w a s s e p a r a t e db y f i l t r a t i o n a n d
d r i e d o v e r K O H a t I t o r r o v e r n i g h t . T h e i s o l a t e ds o l i d ( 3 . 2 g ) w a s 8 , 5 %
p u r e b a r i u m o t . - - l - a n r i n o p r o p a 1n ,e2-- d i o l - 3 - p h o s p h a( 8
t e. 8 m m o l , 4 4 %
r e a c t i o ny i e l d ) a s d e t e r m i n e db y 'q u a n t i t a t i v e3 r PN M R , a n d c o n t a i n e d
45%,o-3-aminopropaneI .2-diol-3-phosphate
as determinedby enzymatic
a s s a y :r F {N M R ( D , o . p H - 1 2 ) 6 ( D S S ) 3 . 7 - 3 . 8( m , I H ) , 3 . 5 0 - 3 . 6 5
r
'
C
( m , 2 H ) . 2 . 7 _ s - : . 9(-m
s. I H):
NMR (D,O,pH -12) 6(DSS)76.6
( d , r " r = 7 . 8 H z ) , 6 8 . 1 ( s ) . 1 9 I ( d . 2 J = 1 . 9 H z ) ; 3 ' P N M R ( D 2 O ,p H
- 1 2 ) a 9 . 1( r , ] J = 8 . 1H z ) .
o - 3 - A m i n o p r o p a n e1-. 2 - d i o l 1
- - p h o s p h a t e . o - 3 - C h l o r o p r o p a n eI-, 2 d i o l - l - p h o s p h a tbea r i u m s a l t ( 6 0 0 m g , 1 . 8m m o l ) w a s a d d e dt o a w a t e r
s o l u t i o n( 5 m L ) . a n d c o o l e di n a n i c e b a t h . D o w e x 5 0 W - X 8 w a s a d d e d
u n t i l t h e o r g a n i c p h o s p h a t ed i s s o l v e d( p H < 2 ) . T h e D o w e x 5 0 W - X 8
beadsand the faint yellow color of the solutionwere removedby passing
t h e s o l u t i o nt h r o u g h c h a r c o a l . T h e D o w e x 5 0 W - X 8 r e s i n w a s w a s h e d
b y p a s s i n ga n a d d i t i o n a l 2 0 m L o f w a t e r t h r o u g h t h e c h a r c o a l . T h e
o-3-chloropropane
1 -. 2 - d i o l --l p h o s p h a t es o l u t i o nw a s a d d e dd r o p w i s et o
a stirred solutionof ammonium hydroxide(25 mL) placedin an ice bath.
After the end of this addition the solutionwas stirred for 30 min at 0 oC,
t h e i c e b a t h w a s r e m o v e d ,a n d t h e r e a c t i o ns o l u t i o nw a s s t i r r e d f o r I h
at ambient temperature. The excessammonia and water were removed
b y e v a p o r a t i o na t 4 0 - 5 0 o C a n d t h e r e s u l t i n go i l w a s d i s s o l v e di n 2 m L
o f w a t e r . B a r i u m c h l o r i d eQ . a g , I . 8 m m o l ) a n d e t h a n o l( 2 0 m L . 9 5 % )
w e r e a d d e d t o p r e c i p i t a t et h e b a r i u m o - 3 - c h l o r o p r o p a n e - 1 . 2 - d i o l - l p h o s p h a t e .T h e s u s p e n s i ow
n a s s t i r r e df o r 1 5 m i n a t 0 o C , a n d t h e s o l i d
w a s s e p a r a t e db y c e n t r i f u g a t i o n .T h e s u p e r n a t a n tw a s d i s c a r d e d .T h e
s o l i d w a s w a s h e dw i t h e t h a n o l ( l 0 m L . 9 5 % , \ .a n d a f t e r c e n t r i f u s a t i o n
7018
J. Am. Chem. Soc., Vol. 107, No. 24, 1985
the solid was dried over CaSOaat I torr overnight. The resultingbarium
o - 3 - a m i n o p r o p a n e1-. 2 - d i o l 1- - p h o s p h a t e( 0 . 5 0 g , 9 4 V o )w a s m o r e t h a n
957o pure as determinedby quantitative3rP NMR (no other phosphate-containing
c o m p o u n dw a s o b s e r v e d ) :r 3 C N M R ( D u O , p H 1 3 ) 6
( D S S ) 7 6 . 8( d . r / = 5 . 5 H z ) , 7 0 . 5 ( d , 2 J = 3 . 7 H z ) , 4 7 . 6 ( s ) ; 3 r PN M R
( D : O . p H l 0 ) 6 - 1 . 5( 1 . U = 6 . 3 H z ) .
A s i m i l a r p r e p a r a t i o nw a s c a r r i e d o u t f o r o l - 3 - c h l o r o p r o p a n e - 1 , 2 d i o l - l - p h o s p h a t eo n a l - m m o l s c a l e . T h e i s o l a t e do l - 3 - a m i n o p r o p a n e 1 , 2 - d i o l - l - p h o s p h a tbea r i u m s a l t ( 0 . 2 8 g ) w a s m o r e t h a n 9 5 % p u r e ( n o
o t h e r p h o s p h a t e - c o n t a i n i ncgo m p o u n dw a s o b s e r v e d )a s d e t e r m i n e db y
q u a n t i t a t i v e3 r P N M R ( 8 8 % r e a c t i o ny i e l d ) .
ot--S-Acetyl-3-mercaptopropane1,2-diol. ol-3-Mercaptopropane1 , 2 - d i o (l l . l g , 1 0 m m o l ) w a s d i s s o l v e idn w a t e r ( 2 5 m L ) a n d c o o l e di n
a n i c e b a t h . T h e s o l u t i o nw a s s t i r r e d f o r 3 0 m i n a t 0 o C a n d a c e t i c
anhydride(2.8 mL, 30 mmol) was slowly added to the solution. The
m i x t u r e w a s s t i r r e d l o r 2 h a t 0 o C b e f o r et h e w a t e r , a c e t i ca c i d , a n d
r e s i d u a la c e t i c a n h y d r i d ew e r e r e m o v e db y e v a p o r a t i o na t I t o r r . ( T h e
t e m p e r a t u r eo f t h e s o l u t i o nm u s t n o t e x c e e d2 0 o C d u r i n g t h e s em a n i p u l a t i o n ss i n c ea c e t v lm i g r a t i o no c c u r sv e r l r e a d i h ' . ) T h e c o l o r l e s sl i q u i d
( 0 . 6 5 g , 4 3 % r e a c t i o ny i e l d ) w a s u s e dw i t h o u t f u r t h e r p u r i f i c a t i o n s : I R
r H N M R ( a c e t o n e - d uD) 3 . 6 - 3 . 7 ( m , I H ) ,
(neat) 1690(S-C:O);
3 . 4 - 3 . 5( m , 2 H ) , 2 . 8 5 - 3 . 1( m , 2 H ) , 2 . 3 ( s , 3 H ) ; r 3 CN M R ( a c e t o n e - d 6 )
1 9 6 . 1( s ) ,7 0 . 4( s ) , 6 4 . 3( s ) ,3 1 . 5( s ) , 2 9 . 9( s ) . T h e ' H N M R a n d I R d a t a
correspondto thoseof o-S-acetyl-3-mercaptopropaneI,2-diol prepared
from o- 1,2-isopropylidene-3-S-acetyl-3-mercaptopropane1,2-diol.57On
oC
storageat 4
the acetyl group migratesfrom sulfur to oxygenover the
courseof 2 months. No thioester bond was observedat the end of this
time.
ol-f/-Methyl-3-aminopropane-1,2-diol Hydrobromide. The DLmethyl-3-aminopropane1,2-diolhydrobromidewas preparedby a modi f i c a t i o n o f t h e p r o c e d u r eo f K n o r r a n d K n o r r . 5 8 o r . - 3 - B r o m o p r o p a n e 1 , 2 - d i o (l 0 . a 5g , 3 . 2 m m o l ) w a s d i s s o l v e idn e t h a n o l( 5 m L . 9 - s ? ) . T h e
e t h a n o l m i x t u r e w a s s l o w l y a d d e d t o a v i g o r o u s l r s t i r r e d s o l u t i o no f
( 4 0 4 . . 5 . 0 g )u ' h i c hh a d b e e np l a c e di n a n i c e b a t h .
a q u e o u sm e t h y l a m i n e
A f t e r l 5 m i n o f s t i r r i n ga t 0 o C . t h e s o l u t i o nu a s h e a t e di o r - i h a t ; 1 0 - 5 0
o C . T h e s o l v e n ta n d e x c e s sn r e t h r l a m i n e\ \ e r c r e m o v e db 1 r o t a r r
e v a p o r a t i o n . R e s i d u a lv o l a t i l e s\ \ ' e r er e m o v e da t I t o r r o v e r K O H r e s u l t i n g i n 0 . 3 5 g o f c o l o r l e s so i l . T h e o r - , \ - m e t h y l - 3 - a m i n o p r o p a n e 1 , 2 - d i o lh y d r o b r o m i d cw a s u s e dw i t h o u t f u r t h e r p u r i f i c a t i o n : r H N M R
( D z O ) 6 ( D S S ) 3 . 9 - 4 . 2( m , I H ) , 3 . 6 - 3 . 7( m , 2 H ) , 3 . 1 - 2 . 3( m . 2 H ) ,
2 . 7 2( s , 3 H ) .
DL-N,lg-Dimethyl-3-aminopropane-1,2-diolhydrobromide was prep a r c d b y a m o d i f i c a t i o no f t h e p r o c e d u r eo f K n o r r a n d K n o r r . 5 8 o r - - 3 B r o m o p r o p a n e - 1 , 2 - d i(o0l. 5 9 g , 3 . 8 m m o l ) w a s d i s s o l v e d
i n e t h a n o l( 5
m L ) . T h e e t h a n o ls o l u t i o nw a s a d d e dt o a s t i r r e ds o l u t i o no l a q u e o u s
d i m e t h l l a m i n c( 5 . 0 g ) . T h e m i x t u r eu a s h e a t e df o r 6 h a t : 1 0 5 0 o C
T h e s o l r e n ta n d e x c e s sd i n r e t h r l a m i n eu c r e r e n t o v e db r r o t a r r c v a p o r a t i o n .a n d r e s i d u arlo l a t i l e s\ \ e r er e m o v e da t I t o r r o v e rK O H o \ e r n i g h t .
T h e c r u d e o t - . \ . \ - d i m e t h rl - - l - a m i n o p r o p a nIe. -l - d i o l h r d r o b r o n t i d e
( y ' e l l o uo i l . 0 . - 1 .g1) u a s u s e d* i t h o u t f u r t h e rp u r i f i c a t i o n l ' l \ \ 1 R
( D 2 O )6 ( D S S )- 1 . 7 - 3 1 (. 9
m . I H ) . 3 . 3 - 3 . , (1m . 2 H ) . 2 . 8 - : . 9 ( n i . I H ) .
2 . 5 5 ( s , 6 H ) . T h e s p e c t r a ld a t a a r e i n a c c o r d w i t h t h o s ep r e r i o u s l r
published.5e
o l - N - A c e t y l - 3 - a m i n o p r o p a n e - 1 , 2 - d i ool .t - - 3 - A m i n o p r o p a n e1-, 2 - d i o l
( 9 . 1 g , 1 0 0 m m o l ) w a s d i s s o l v e di n a c e t i ca n h y d r i d e( 2 5 m L ) a n d s t i r r e d
overnightat ambient temperature. The acetic anhydride and acetic acid
w e r e r e m o v e db y r o t a r y e v a p o r a t i o na. n d t h e r e s u l t i n go i l w a s d i s s o l v e d
i n m e t h a n o l . S o l i d o o t a s s i u mc a r b o n a t ew a s a d d e d t o t h e m e t h a n o l
solutionand the suspensionwas stirred for 2 days. After the solventwas
r e m o v e d b y ' r o t a r l e v a p o r a t i o n ,t h e l i q u i d c o n t a i n e d o l - A - a c e t y l - 3 a m i n o p r o p a n e - 1 , 2 - d i or el ,s i d u a ls o d i u ma c e t a t e a
, n d m e t h a n o l( t o t a l 9 . 5
g ) . T h i s m i x t u r e c o n t a i n i n go t - - ' V - a c e t y l - 3 - a m i n o p r o p a n
1e
, 2- - d i o lw a s
u s e dw i t h o u t l u r t h e r p u r i f i c a t i o n :' H N M R ( D z O ) D ( D S S ) 3 . 6 3 - 3 . 7 3
( m , I H ) , 3 . 3 s - 3 . 5( m . 2 H ) , 3 . 0 5 - 3 . 2 5( m , 2 H ) , 1 . 9( s , 3 H ) ; r 3 CN M R
( D z O )6 ( D S S ) 1 7 8 . 6( s ) , 7 4 . 6( s ) , 6 7 . 7( s ) , 4 6 . 2( s ) , 2 6 . 3( s ) .
ol-GlyceraldehydeDimethyl Acetal was preparedby osmium tetroxide
catalyzed oxidation of acroleindimethyl acetal.60 N-Methylmorpholine
1 V - o x i d e( 1 a . 8 g , 1 0 7 m m o l ) a n d o s m i u m t e t r o x i d e ( 7 0 m g ) w e r e d i s s o l v e di n a m i x t u r e o f w a t e r ( 4 0 m L ) a n d a c e t o n e( 2 0 m L ) . T h e r e a c t i o n
( 5 7 ) G r o n o w i t zS, . ; H e r s l o f ,B . ; M i c h e l s e nP, . ; A a k e s s o n , BC. h e m .P h y s .
Lipids 1978,22, 307-21.
( 5 8 ) K n o r r ,L . ; K n o r r ,E . C h e m .B e r . 1 8 9 9 , 3 2 , 7 5 0 - 7 .
( 5 9 ) P o u c h e r tC
, . J . * T h e A l d r i c h L i b r a r y o f N M R S p e c t r a " ,2 n d e d . ;
A l d r i c hC h e m i c aC
l o . : M i l w a u k e eW
, i., 1984.
( 6 0 ) V a n R h e e n eV
n ,. : C h a ,D . Y . ; H a r t l e y W
, . M . I n " O r g a n i cS y n t h e s e s " ;
S h e p p a r dW
, . A . , E d . ;W i l e y : N e w Y o r k . 1 9 7 8 ;V o l . 5 8 , p p 4 3 - 5 2 .
Crans and Whitesides
m i x t u r e w a s p l a c e di n a n i c e b a t h . A c r o l e i n d i m e t h y l a c e t a l ( l 1 . 8 m L ,
100 mmol) was added to the solution,and the mixture was slowly
e q u i l i b r a t e dt o a m b i e n t t e m p e r a t u r e( 4 - 6 h ) . T h e s o l u t i o nw a s s t i r r e d
overnight. Sodium hydrosulfite(0.5 g) and magnesiumsilicate(Woelm,
Eschwege,6 g) were addedto the black solution,and the suspenslonwas
stirred for 20 min beforethe solidswere removedby filtration. This step
w a s r e p e a t e du n t i l a l l o s m i u m e s t e r sw e r e r e m o v e da n d t h e f i l t r a t e w a s
r e d b r o w n . T h e s o l v e n t a n d N - m e t h y l m o r p h o l i n ew e r e r e m o v e d b y
evaporation. The residualvolatileswere removedovernightover CaSOo
a t I t o r r ; a c o l o r l e s sl i q u i d ( 9 . 6 g ) r e s u l t e d . T h e l i q u i d c o n t a i n e dN methylmorpholine(-25Vo), but was usedwithout further purification to
test substrateactivity with glycerolkinase: rH NMR (DzO) d (DSS) 4.3
( d ," l = 6 . 0H z ) , 3 . 6 5 - 3 . 7 0
( m , 3 H ) , 3 . 6 1( s , 3 H ) , 3 . 6 2 ( s , 3 H ) ; ' 3 C
N M R ( D z O ) d ( D S S ) 1 0 9 . 3( s ) , 7 5 . 8 ( s ) , 6 6 . 4 ( s ) , 6 0 . 2 ( s ) , 5 9 . 7 ( s )
( 1 V - m e t h y l m o r p h o l i rnreC; N M R ( D : O ) 6 ( D S S ) 6 9 . 1 ( s ) , 5 7 . 9( s ) , 4 8 . 2
( s ) ) ; m a s ss p e c t r u m m
, lz 136 (M), 135(M - 1), 104(M - CH2OH(ocHr)). t0s (M - I - cH2oH(ocH3)), 75 (M - CH2OHCHOH), 3l
( C H T O H ) . T h e r H N M R s p e c t r u mi s i n a c c o r dw i t h a p a r t i a l s p e c t r u m
p u b l i s h e dp r e v i o u s l y . 6 r
o r - 3 - A m i n o - l - c h l o r o p r o p a n - 2 - o lH y d r o c h l o r i d e . n l - 3 - A m i n o - l c h l o r o p r o p a n - 2 - owl a s p r e p a r e db y a c i d i c h y d r o l y s i so f l - b e n z a l i m i n o 3 - c h l o r o p r o p a n - 2 - oal c c o r d i n gt o t h e p r o c e d u r eo f P a u l e t a l . : 6 2 m p
l 0 l - 1 0 2 " c i l i t . 6 2l 0 l - 1 0 2 " c l ; ' H N M R ( D 2 o , p H 7 ) 6 ( D S S ) 3 . 9 - 4 . 2
( m , 1 H ) , 3 . 5 0 - 3 . 6 5( m , 2 H ) 8 3 . 0 - 3 . 2( m , 2 H ) : r r C N M R ( D 2 O , p H
7) 6 (DSS) 71.7(s), sO-ss (s), 46.6(s)
o r - 3 . 4 - D i h y d r o x y b u t a n o n i t r i l ew a s p r e p a r e d f r o m o t - - 3 - c h l o r o propane-l .2-diol by nucleophilicsubstitutionof the chloride with sodium
c y a n i d e . 6 r o t - J - C h l o r o p r o p a n e1-, 2 - d i o l( 2 2 g , 2 0 m m o l ) w a s d i s s o l v e d
i n w a t e r ( - r m t ) a n d c o o l e di n a n i c e b a t h . S o d i u m c y a n i d e( 1 . 0 g , 2 l
m m o l ) w ' a sa d d e d t o t h e s o l u t i o n a n d s t i r r e d f o r 3 h a t 0 o C . T h e
r c a c t i o n m l x t u r e w a s p a s s e dt h r o u g h a n i o n - e x c h a n g ec o l u m n ( A G
- i 0 l \ \ ' - X 8 . 6 0 g o f ' r e s i n )a n d w a s h e dw i t h i c e - c o l dm e t h a n o l( 1 0 0 m L ) .
T h e c o r n b i n c df r a c t i o n sw e r e e v a p o r a t e dt o d r y n e s s . T h e r e s u l t i n go i l
( l - 1 g ) u a s u s c du ' i t h o u tf u r t h e r p u r i f i c a t i o n :I R ( n e a t ) 2 2 6 0 ( C N ) ; r H
\ \ { R ( D . O ) , ( D S S ) 3 . 2 - 3 . 6( m , 3 H ) , 2 . 5 - 2 . 7( m , 2 H ) : I r C N M R
( D r O ) ( D S S ) 1 2 3 . 9( s ) .7 1 . 9( s ) ,6 8 . 7( s ) ,2 6 . 4( s ) ;m a s ss p e c t r u mm, f z
1 0 2( M + 1 ) . l 0 l ( M ) , 8 4 ( M + r - H 2 O ) , 7 0 ( 7 2 )( M + I - C H 2 O H ) ,
6 r ( M - C H 2 C N ) ,4 l ( M + r - C H O H C H 2 O H ) , 3 1 ( C H 2 O H ) . T h e
s p e c t r a ld a t a c o r r e s p o n dt o t h o s e r e p o r t e db y J u n g a n d S h a w . 6 a
o t - - 3 - A m i n o - l - m e t h o x y p r o p a n - 2 -H
o ly d r o c h l o r i d e . n l - 3 - A m i n o - l methoxypropan-2-olwas prepared in two steps. First, acid-catalyzed
m e t h a n o l y s i so f e p i c h l o r o h y d r i nf o r m e d o t - - 3 - c h l o r oI-- m e t h o x y p r o p a n 2 - o l . 6 s S e c o n d .n u c l e o p h i l i cd i s p l a c e m e not f t h e c h l o r i d eb y a m m o n i a
p r o d u c e do t - -3 - a m i n o -I - m e t h o x y p r o p a n - 2 - o lo. t - -E p i c h l o r o h y d r i n( 0 . 93
g . l 0 m m o l ) u a s d i s s o l v e idn d r y m e t h a n o l( 1 0 m L ) , a n d c a t i o n - e x c h a n g e
r c s i n ( D o * ' c r 5 0 W - X 8 . 1 0 0m g ) w a s a d d e dt o t h e m i x t u r e . T h e s o l u t i o n
u r r s h c a t e da t - \ 0 o C f o r 1 , 5h . T h e s o l u t i o nw a s f i l t e r e d t o r e m o v et h e
c a t i ( ) n - c \ c h a n gree s i n ,a n d t h e s o l v e n tw a s r e m o v e db y e v a p o r a t i o n .T h e
r c s u l t i n go i l w a s d i s s o l v e idn w a t e r ( 5 m L ) a n d t h i s m i x t u r e w a s a d d e d
d r o p ui s et o a n i c e - c o l ds t i r r e da q u e o u ss o l u t i o no f a m m o n i u mh y d r o x i d e
t l 0 m l . l ; 1 \ { ) w h i c h h a d b e e np l a c e di n a n i c e b a t h . T h e r e a c t i o nw a s
s t i r r c d a t 0 o ( - l ' o r I h . A f t e r e v a p o r a t i o no f t h e s o l v e n ta n d w a s h i n go f
t h c s o l i d i r i t h e t h e r .0 . 6 g ( a s a c o l o r l e s so i l ) o i c r u d e o r - 3 - a m i n o - l n r e t h o r r p r o p a n - 2 - ohl y d r o c h l o r i d er e s u l t e d . T h e c o m p o u n dw a s u s e d
w i t h o u tf u r t h c rp u r i f i c a t i o n :r H N M R ( D : O . p H 7 ) 6 ( D S S ) 1 . 0 - 4 . 2
( m , I H ) . 3 . 5 - - 1 .( 6m . 2 H ) , 3 . 4 ( s , 3 H ) . 3 . 1 - - r . (3m , 2 H ) : ' ' C N M R
( D 2 O ,p H 7 ) 6 ( D S S ) 7 8 . 3 ( s ) , 7 0 . 5 ( s ) . 6 3 . 3 ( s ) , 4 6 . 5 ( s ) ; m a s ss p e c t r u m ,
m f z 1 0 6( M + l ) , 1 0 5( M ) , 8 8 ( M + I - H r O ) , 8 7 ( M - H : O ) .
was preparedaccordingto the procedure
2-Methylpropane-1,2,3-triol
o f E i s e n t h ael r a l . : 2 4r H N M R ( D : O ) 6 ( D S S ) 3 . 3 ( s , 4 H ) . 0 . 7 ( s . 3 H ) .
2-(Hydroxymethyl)butane-1,2-diol (2-Ethylpropane-1,2,3-triol) was
p r e p a r e da c c o r d i n gt o t h e p r o c e d u r eo f E i s e n t h a le t a l . : l a l t l N M R
( D z O ) 6 ( D S S ) 3 . 4 ( s . 4 H ) , 1 . 3( q , 2 H ) . I I ( t , 3 H ) ; m a s ss p e c t r u m ,
m l z t l 7 ( M - 3 ) . 1 0 3 ( M - O H ) , 9 l ( M - C H 2 C H 3 ) , 8 e( M c H 2 o H ) , 4 3 ( M - O H - C H 2 O H - C H 2 C H T ) ,3 l ( C H 2 O I { ) .
Supplementary Material Available: Tables of Vm ^ and K* for
a number of substrates for glycerol kinase for C. mycoderma and
B. stearothermophilus (2 pages). Ordering information is given
on any current masthead page.
( 6 1 ) C a p o n ,B . ; T h a c h e rD
, . J . C h e m .S o c . 8 1 9 6 7 . 1 3 2 2 - 6 .
( 6 2 ) P a u l ,R . : W i l l i a m s ,R . P . ;C o h e n E
, . J . O r g .C h e m . 1 9 7 5 , 4 0 . 1 6 5 3 - 6 .
( 6 3 ) K o e l s c hC, . F . J . A m . C h e m .S o c . 1 9 4 3 , 6 5 , 2 4 6 0 - 5 .
( 6 4 ) J u n g ,M . E . : S h a w ,T . J . J . A m . C h e m .S o c . 1 9 8 0 ,1 0 2 . 6 3 0 4 - l l .
( 6 5 ) F o u r n e a uE
. , . ;R i b a s ,L B u l l . S o c .C h i m . F r . 1 9 2 6 . 1 5 8 4 - 9 .