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and H Z SableD S Shreve, M P Holloway, J C Haggerty, 3rddehydrogenase are not identical.states for transketolase and pyruvateThiamin pyrophosphate-derived transitionThe catalytic mechanism of transketolase.:
1983, 258:12405-12408.J. Biol. Chem.
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THE
OURNAL
F BIOLOGICALHEMISTRY
Vol.
258,
No. 20, Issue of October 25 pp. 12405-12408,1983
Printed in U S A
The Catalytic Mechanism f Transketolase
T H I A M I N P Y R O P H O S P H A T E - D E R I V E D T R A N S I T I O N S T A T E S F O R T R A N S K E T O L A S E A N D P Y R U V A T E
D E H Y D R O G E N A S E A R E N O T I D E N T I C A L *
(Received for publication, February 17, 1983)
David
S.
ShreveS, Michael
P.
Holloway, Jesse
C.
Haggerty,
1118,
and Henry
2
Sable7
From the Department
o
Biochemistry Case Western Reserve University Cleveland Ohio 441 6
Thiamin thiazolone pyrophosphate (TTPP) hasbeen
reported to be an effective transition state analogue
for the thiamin pyrophosphate-dependent partial re-
action of pyruvate dehydrogenase (Gutowski, J. A.,
and Lienhard,G. E.
1976) Biol Chem 251,2863-
2866).The kinetics of the interaction of TTPP with
transketolase are reported here. TTPP is
a
competitive
inhibitor, with respect to thiamin pyrophosphate, of
bakers yeast transketolase but it is neither a tight
binding inhibitor nor
a
slow binding inhibitor. TTPP
decreases the kinetically observed negative coopera-
tivity seen for thiamin pyrophosphate and also de-
creases the ra te constant for the hysteretic activation
of the enzyme by thiamin pyrophosphate. We conclude
that thiamin thiazolone pyrophosphate is not an effec-
tive transition tate analogue for the eaction catalyzed
by bakers yeast transketolase.This difference be-
tween transketolase and pyruvate ehydrogenase may
be related to differences in the polarity of the active
sites
of
the enzymes. It is conceivable that the active
site of the pyruvate ecarboxylase subunit
of
pyruvate
dehydrogenase is hydrophobic, by analogy with the
known hydrophobicity of the active site of brewers
yeast pyruvate decarboxylase. This hydrophobicity
would stabilize a transition tate with no charge on the
thiazole portion of the coenzyme, similar to the un-
charged thiazole portion of TTPP. In contrast, the
active site of bakers yeast transketolase, which is
known to containcharged amino acid side chains,
should be less favorable forsuch an uncharged transi-
tion state. A charge-separated canonical form related
to TTP P could be referentially stabilized in the active
site of transketolase.
Transketolase transfers a keto1 group from a donor mole-
cule (a ketose pho sph ate) to an accep tor molecule ( an aldose
~
Grants AM-18888,
5-T32-GM-07225,
and 5-T32-AM-07319.
This is
*T hi s research
was supported by
National Institutes of
Health
paper VI in the series Enzymes
of
Pentose Biosynthesis. For paper
V,
see Egan and Sable (2).
A
preliminary report has been published
25).
The costsof publication of this article were defrayed in par tby
the payment of page charges. This article must therefore be hereby
marked aduertisement in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Present address: The Goodyear Tire and Rubber Company, Re-
search Division, 142 Goodyear Blvd., Akron, OH 44316.
Clifton Rd., N.E., Atlanta, GA 30322.
3
Present address:EmoryUniversity School of Medicine, 1440
fi
To whom correspondence and requests for reprints should be
addressed.
phospha te )
1)
nd requi res th iamin-PP anddivalent cation
for ac t iv i ty . Thiamin-PPlowly act ivates the inact ive apoen-
zyme, and he ime required toreach V, dependson he
concentrat ion of th iamin-PP (2). Gut owsk iandLienhard
synthesized TTPP (see Fig. 1) and reported tha t i t inact i-
vates pyruv ate dehydrogenase from Escherichia coli with K
0.5 nM
3) .
The y also reported that the affini ty of
E.
coli
pyruvate dehydrogenase is a t least 2
X
lo4 imes greater for
TTPP tha n for th iamin-PP and tha t the ha l f - t ime for the
release of
TTPP
from the enzyme is
40
h. These results and
their knowledge
of
the mechanism of catalysis of decarbox-
ylation of pyruvate led them o propose th at TTPP is a
transi t ionstateanalogue for pyruvate dehydrogenase an d
should be a potent inhibi tor for other thiam in-PP requ iring
enzymes. Butler
et
al.
(4 )
have eported hat
TTPP
is a
trans ition state analogue also for the first partial reaction
catalyzed by bovine kidney pyruv ate dehydrogenase. Th ese
results and the slow act ivat ion of t ransketolase by thiamin-
PP suggested that TTPP might be a slow, tightbinding,
transitio n state analo gue for transketolase. In this p aper, we
report studiesof th e effects of
TTPP
on the ra te cons tantor
act ivat ion of t ransketolase by thiamin-PP 7 - l ) and on Vss.
The resul ts show tha t
TTPP
is not an effective transi t ion
state analogue
for
transketolase.
MATERIALS A N D METHODS
R E S U L T S
The l inea r dependencef V, on the concentrat ion f t rans-
ketolase mono mer, both in theresence and abse nce f
TTPP
(Fig. a , shows tha t TTPP is n ot a tight binding inhibitor of
transketolase (18).The resul t s shown inFig.
3
prove that V,,,
is un affected by the presence
or
absence of TTPP; th i s indi -
ca tes tha t
TTPP
is
a
competitive inhibitor of transketolase
with respect to th iam in- PP . Negative cooperativity with re-
spe c t t o t h i a mi n -P P as rep orted previously (2 ) a nd t he s a me
The abbreviations used are: thiamin-PP, thiaminpyrophosphate;
Rib-5-P, ribose 5 - P
S0.5
the concentration
of
thiamin-PP at which
V,, =
0.5
V,,,;
TTPP, thiamin thiazolonepyrophosphate;
V,,
the
maximum, steady state velocity attained with saturating concentra-
tions of thiamin-PP, M e , Rib 5-P, and Xlu-5-P;
V,,
steady state
velocity: XIu-5-P, xylulose 5-P
D-threo-2-pentdose-5-P).
* Portions of this paper (including Materials and Methods, Figs.
1-6, and Table
I)
are presented in miniprint t the endf this paper.
Miniprint is easily read with the aid of a standard magnifying glass.
Full size photocopies are available from the Journal of Biological
Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Doc-
ument No. 83M-391, cite the authors, and include a check or money
order for 4.40 per set of photocopies. Full size photocopies are also
included in the microfilm edition of the Journal thats available from
Waverly Press.
12405
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12406
Transi t ion State Transhetolase
phenomenon s observed both n he presence and n he
absence of TT P P,
as
show n in Fig. 3.
TTPP (160 nM) de crease d the negative cooperativity seen
for thiamin-P P b inding (Fig.
4).
The slopes of H il l plots for
exper iments inwhich TTPP was abse nt were 0.6 .1 n=
2). This result agrees well with values previously obtained of
0.61 (2) and
0.59.3
T h e slopes of Hill plots for assay m ixtures
conta ining 160 nM
TTPP
we re 0.87 0.02 n=
4).
So for
th iamin-PP, in the absence
f
TTPP, was
3.48 0.04
p~
n
= 2), ingood agreement witha previously o btained value of 2
p ~ . ~n the presenceof 160 nM TTP P , So 5was higher, 6.8
0.9
p M
n=
4 ) .
Fig. 5 shows a plot of 7- l
versus TTPP
concentrat ion for
an experimen t in which the react ion was ini t ia ted with di-
meric apoenzyme. At co ncentrat ions 2120 pg/ml, the apoen-
zyme is dimeric, butat lower concentrat ions here is an
equilibriumbetween monomeranddimer 14) .Reactions
ini t ia ted with enzyme solut ions containing40
or
50 pg/ml of
transketolase gave results sim ilar to th ose sh own inig.
5
in
t h a t 7 - l decreased with increasingTTPP concentrat ion. This
indica tes tha t
TTPP
decre ases the rate of activation when
the assay is ini t ia ted with dimer ic apoenzyme as well as in
those ni t ia tedwi th
a
mixture of monomeric and dimeric
apoenzyme. Values of V obtained in the exp eriment shown
in Fig. 5 were used to calcu late K, for TTPP from a Dixon
plot (17) (Fig. 6). The data in Fig. 6 give a value of Ki
26
nM. In other experim ents in w hich 40
or 50
pg/ml of trans-
ketolase were used to initiate the assays, the
Ki
as 37 a n d
22 nM, respectively.
Table
I
shows the effects on
V8,
a n d
7 l
of incubation of
apotransketolase with Mg -TTPP prior o assay. When di-
mericapoenzymewas incuba ted wi th Mg-T TPP and hen
assayed in the prese nce of the same concentrat ion of Mg-
TTPP
a n d
1 p~
thiam in-PP , the values of V,, a n d
7-l
did
not differsignificantly from hose observedwhendimeric
apotransketolase, ncubated n he absence of T TP P, was
assayed under the same co ndit ions. Co ntrol studies, inhich
the assay mixture conta ined no
TTPP
and the enzyme had
not been incubated with TTPP, showed that , un der th e con -
ditions used, TTPP inhibi ted the enzyme an d caused a de-
crease n 7-l. The fa c t ha t 7-l is not decreased by prior
incubation of theenzymewi thMg-TTP P ndica tes ha t
TTPP
is not a slow binding inhibitor of transketo lase
(18).
DI SCUSSI ON
The purpose of this study was to invest igate the inhibi t ion
of bakers yeast ransketolas e by
TTPP
and the effect of
TTPP
on
7-l
for the act ivat ion of t ransketo lase by thiamin-
PP. Fig. 2 shows the TTPP is not
a
tight binding inhibitor.
In this re spect , t ransk etolase iffers from the pyruvate dehy-
drogenase of E . coli
3 )
an d bovine kidney
4) .
In thos e cases,
TTPP is a t ransi t ion ta te analogue and nactiva tes he
enzym e alm ost irreversibly. Fig.
3
shows tha t TPPP is com-
peti t ive with respect to th iam in-P P, a nd the Hil l plo ts Fig.
4)
in the presence and absen ce of
TTPP
showed tha t
TTPP
partially abolishes the negative cooperativity seen with thia-
min-PP. The effect of TTPP on the cooperativity could be
due to the format ionf t ransketolase dimers n which
TTPP
i s bound to one subuni t and th iamin-PP i s bound to the o the r
subu nit . The negative cooperat ivi ty would bedecreased if
TTPP binding did not inhibi t the b inding of th iamin -PP to
the o the r subuni t . Thiamin-PP b inding to the f i rs t subuni t
does cause su ch inhib ition, as indica tedy th e negative coop-
J. C. Haggerty,
111,
D. s.Shreve, and H. Z. Sable,
1983) Comput.
Biol.
M e d.
submitted for publication.
erativity seen inFig.
3.
This explana t ion assumes tha t t rans-
ketolase exhibi ts t rue s i te-si te interact ions throughpace an d
that the coop erat ivi ty een with transketolase is not a result
of the hysteresis
19).
Fig. 5 shows tha t T- decreases with increasing concentra-
tions of
TTPP.
This result was obtained whether react ions
were ini t ia ted with dimer
or
with a mixture of monomer an d
dimer. The data of Egan and Sable
(2)
indicate that dimeri-
zation of inactive enzym e subun its may be one
of
the slow
steps in the act ivat ion of th e enzyme by thiamin- PP. Other
possible slow steps could involve slow binding of thia mi n-P P
or a slow conformationa l change in the enzyme, other than
dimerizat ion. The decrease in -l with increasing concentra-
tions of TTPP could be due to decrease in the rate consta nt
for some,as yet ndefined, slow step in the act ivat ionrocess.
Alternat ively, the decrease in 7-l could be due to a decrease
in the concent ra t ionof the enzy me form tha t undergoes the
slow step. The inhibi tor could divert the enzyme into dead-
end, nhibi tor-enzyme complexes that do not undergo the
slow conversion that is necessary for the activation. Studies
now in progress4 on hemec han ism of activation of th e
enzyme by th iam in- PP may yield inform ation that wil l help
us to und erstand the influence of these and other inhibi tors
on
7- l .
Exp erim ents in which dimeric a potransk etolase was incu-
bated ei ther with
or
without TTPP and then assayed in the
presence or a bsence of TTPP (Table I showed tha t TTPP is
no t a slow binding inhibitor of transketolase. K of TTPP
with transk etolase is 28
*
nM in reactions initiated either
with dimeric apotransketolase or with m ixtures of dim er and
monomer. This is nearlywo orders of magn itude greater than
the est im ated u pper l imit f
0.5
nM
for
t he Ki of
TTPP
with
pyruv ate dehydrogenase
3).
For oxythiamin-PP(II), known
competitive inhib itor of transketolase,
K =
32 nM was found
in reactions initiated withd ime ri c a po t ra n~ ke t o l a se .~
Gutowski and Lienhard,who first synthesized T TP P, con-
sidered i t to be
a
trans ition state analogue resembling th e
proposedncharged,etastablenaminentermediate
formed durin g the decarboxylation of pyruvate by pyruva te
dehydrogenase or pyruv ate decarboxylase
3).
It seemed rea-
sonable to postulate a re lated enamine intermediate
as
t he
t rans i t ion s ta te dur ing the t ransfe r f the keto1 group in the
transketolase react ion 1, 20). In the case of pyruvate dehy-
drogenase, the interm ediate is the enam ine form of the
a-
carbanion of
2-(a-hydroxyethyl)thiamin-PP
111); n th e case
of t ransketolase, t should be the enam ine form of the a-
carbanion of
2-(a,P-dihydroxyethyl)thiamin-PP
V).Despite
the superfic ial similari ty between the enamine intermediates
in the wo react ions, TTPP binds much less t ightly to t rans-
ke tolase than to thedehydrogenase;
Ki
for TTPP (28 nM) is
anorde r of magn itude ess han he lowest K for Mg-
t h i a m i n - P P
(0.4p ~ )
2). The replacemen t f the methyl roup
of 111 by an hydroxy methyl group in
V
results in a marked
increase in the polari tyf V relative to
111.
AS a consequence,
TTPP is less appropriate as a t ransi t ion sta te analogue for
the transketolase react ion than the pyruvate decarboxylase
reaction. T his is reflected in its b indin g m uch less tightly to
transk etolase han o pyru vate dehydrogenase. The differ-
ences in the affini tyf TTPP for t ransketolase and pyruvate
dehydrogenase must reflect considerable differences between
th e active sites of the enzymes. Even hough both require
thi am in- PP an d bo th catalyze mechanist ically similar reac-
t ions, hey use differentsubstrates. Crosby et al. (21,
22
reported that adducts
f
pyruva te wi th th iamin-PP a re decar-
D.
S .
Shreve, J. C. Haggerty, 111, M .
P.
Holloway, and H. Z. Sable,
manuscript in preparation.
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Tran sition State Transketolase 12407
boxylatedmore apidly as he po larit y of the solvent de-
creases , and hey hyp othesiz ed hat he active si tes of a l l
thiamin-PP requ iring enzymes would be fou nd to be hydro-
phobic. In contr ast, the a ctive site of transke tolase h as been
found o contain charged amino acids and s undou btedly
more exposed to the aqueous solvent than is thective s ite of
pyruv ate decarboxylase 23,
24).
The re sults pre sented in this study suggest that no single
s t ruc ture can represent the t rans i t ion s ta tesf all enzymatic
reac t ions in which th iam in-P P is the coenzyme. The differ-
ences amon g he ransi t ion sta tes will reflect a t least he
differences in stru ctu re and degree of polarity of the various
active si tes. The uncharged , enamine structure of I11 is ap-
prop riate for an d could be stabilized by the apo lar env iron-
me nt of the active site
f
pyruv ate decarboxylase. In con tras t,
interact ion between the highly po lar environ ment of the ac-
tive site of transketo lase 23, 24) and the uncharged, subst i-
tuted thiazole port ion of Va would be unfavorable. A more
appropriate formu lat ion f this t ransi t ion sta te is the ch arge-
separated canonical form Vb. Such a t ransi t ion sta te would
be expected to be stabil ized b y comp lementaryharges in the
active si te as well as bydiffusion of th e formal,negative
charge over the oxygen atoms of thea ,P-dihydroxye thyl
group.
W econclude that
TTPP
is a competitive inhibitor for
bakers yeast t ransketolase with respect to thiam in-P P bu t is
not a slow, tight bind ing inhib itor f th is enzyme. Because it
isnot ightbind ing, t does not unction effectively as a
transi t ion sta te analogue for t ransketolase.
REF ERENCES
1.
Racker, E.
(1961)
in The Enzymes (Boyer, P. D., Lardy, H., and
Myrback, K., eds) Vol. 5,
pp.
397-406, Academic Press, New
York
2. Egan, R. M., and Sable, H. Z. (1981) J . Biol. Chem. 25 6, 4877-
4883
SUPPLEMENTARY MT ERI AI
TO
D a v i d 8 S h r e v e . U i c h i l e l
P
H o l l o w d y ,
Jesse C
H a g g e r ty .
I l l
and
Henry 1 Sable
g l y c e r q lp h o s p h a t ed e h f l d r o g e n a s e
L C . 1 1 1 81
and t v i o r e p ho s ph a te
ismerase E.C.
53-111
L y o p h l l l z e d b a k e r s ' y e a s t r a n r k e t o l a r e E.C. 2.2.1.1) ( l o t 3 l F - 8 0 2 5 ) , l y o p h i l i z e d a-
Rib-5-P 11 -5-P and
DPNH
were o b t a i n e d r o m
Slgna.
A l l o t h e r C h e m i ca l s
w e r e Of
r e a g e n t
grade
3.
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12.
13.
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15.
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Gutowski, J. A., and Lienhard, G.
E.
(1976)J . Biol. Chem. 251,
Butler,
J.
R., Pettit, F. H., Davis,
P. F.,
and Reed, L. J.
(1977)
Sykes, P., an d Todd, A. R. (1951)J.
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Soc. Perkin Trans.
Haggerty, J. C., 111
(1982)
Ph.D.Dissertation, Case Western
Gutowski,
J. A. (1979)
Methods Enzymol.
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Berenblum,
I.,
and Chain, E.
(1938)
Biochem. J .
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Gallo, A. A., Ha nse n, I. L., Sable, H. Z., and Swift, T. J. (1972)
Weber,
K.,
and Osborn,
M.
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Ullrich, J., and Mannschreck,
A. (1967)
Eur. J . Biochem.
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Heinrich, C. P., Noack,
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and W iss,
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TABLE
Ef fe c ts an V,- and
7 - l of
P r e i n c u b a t i o n of A p o t r a n r k e t o l a r e mth TTPP
nn
n P P ,
3
n l HgCI2 i n 42
r*l
T r i s - C l , pH
7.6.
C o n t ro l n cu b a t i o n s
i n c l u d e do n l y enzyme. wPCIz and Tr i s-C l . Assays rere th e n e r fa me d
i n h e p r e s e n c e or absence O f 250 nM TTPP as d e sc r i b e d
~n
th e H e th o d l
s e c t i o n .A l l
va l u er re
p re se n te d d l average
t
S D
VsI 1 5 ~n u n i t s
o f AA mn and
i r
i n u n i t s f . in - ' . The th i a m i n -PPo n ce n t ra -
t i o n = 1 VU.
The
p re i n cu b a t i o nm i x tu re s o n ta i n e d
0 . 135
ng/d of
t r a n r k e t o l a r e . r r a y r
we~e
n i t i a t e d i t h
IO
of m i n c u b a t e d
r a l u t i o n
of e n z w .
A l l a s s a ys e r e Perfomred in w a d r u D l i c a t e .
Dimric
a p Ot rd n l ke to l l Oe was I n c u b a t e da t 25
C
fo r h w r h 250
TTPP
SI
In fimt ? n u b a t i o n ~n
absentbsent
0 . 3 4 1
f
0
009
0.56
t
0.07
absentresent
0.213
0.007 0.31 0 .05
Pre se n t 0 .2 0
t
0 01
0.27
f 0.04
7esent
bygue
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