:

The catalytic mechanism of transketolase.

Thiamin pyrophosphate-derived transition
states for transketolase and pyruvate
dehydrogenase are not identical.
D S Shreve, M P Holloway, J C Haggerty, 3rd
and H Z Sable
J. Biol. Chem. 1983, 258:12405-12408.

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THEJOURNAL
OF BIOLOGICAL
CHEMISTRY

Vol. 258, No. 20, Issue of October 25, pp. 12405-12408,1983

Printed in U.S.A.

The Catalytic Mechanismof Transketolase

THIAMIN PYROPHOSPHATE-DERIVED TRANSITION STATES FOR TRANSKETOLASE AND PYRUVATE
DEHYDROGENASE ARE NOT IDENTICAL*
(Received for publication, February 17, 1983)

David S. ShreveS, Michael P. Holloway, Jesse C. Haggerty, 1118, and Henry 2. Sable7
From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

Transketolase transfers a keto1 group from a donor molecule (a ketose phosphate) to an acceptor molecule (an aldose

phosphate) (1)and requires thiamin-PP and

a divalent cation
for activity. Thiamin-PPslowly activates the inactive apoenzyme, and the time required to reach V , dependsonthe
concentration of thiamin-PP (2). GutowskiandLienhard
synthesized TTPP (see Fig. 1) and reported that it inactivates pyruvate dehydrogenase from Escherichia coli with K ,
5 0.5 nM (3). They also reported that the affinity of E. coli
pyruvate dehydrogenase is a t least 2 X lo4 times greater for
TTPP than for thiamin-PP and that the half-time for the
release of TTPP from the enzyme is 40 h. These results and
their knowledge of the mechanism of catalysis of decarboxylation of pyruvate led themto propose that TTPP is a
transitionstateanalogue
for pyruvate dehydrogenase and
should be a potent inhibitor for other thiamin-PP requiring
enzymes. Butler et al. (4) havereportedthat
TTPP is a
transition state analoguealso for the first partial reaction
catalyzed by bovine kidney pyruvate dehydrogenase. These
results and the slow activation of transketolase by thiaminPP suggested that TTPP might be a slow, tightbinding,
transition state analogue for transketolase. In this paper, we
report studiesof the effects of TTPP on the rate constant
for
activation of transketolase by thiamin-PP (7-l) and on Vss.
The results show that TTPP is not an effective transition
state analogue for transketolase.
MATERIALS A N D METHODS
RESULTS

The linear dependenceof V, on the concentrationof transketolase monomer, both in the

presence and absenceof TTPP
(Fig. a), shows that TTPP is not a tight binding inhibitor of
transketolase (18).The results shown inFig. 3 prove that V,,,
is unaffected by the presence or absence of TTPP; this indicates that TTPP is a competitive inhibitor of transketolase
with respect to thiamin-PP. Negative cooperativity with respect to thiamin-PP
was reported previously (2) and the same

*This research was supported by National Institutes of Health

Grants AM-18888, 5-T32-GM-07225, and 5-T32-AM-07319. This is
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 part by
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, Research Division, 142 Goodyear Blvd., Akron, OH 44316.
3 Present address:EmoryUniversity School of Medicine, 1440
Clifton Rd., N.E., Atlanta, GA 30322.
fi To whom correspondence and requests for reprints should be
addressed.

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 concentrations 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 miniprintat the endof 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 Document 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 thatis available from
Waverly Press.

12405

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Thiamin thiazolone pyrophosphate (TTPP) hasbeen

reported to be an effective transition state analogue
for the thiamin pyrophosphate-dependent partial reaction of pyruvate dehydrogenase (Gutowski, J. A.,
and Lienhard, G. E. (1976)J. Biol. Chem. 251,28632866). 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 cooperativity seen for thiamin pyrophosphate and also decreases the rate constant for the hysteretic activation
of the enzyme by thiamin pyrophosphate. We conclude
that thiamin thiazolone pyrophosphate is not an effective transitionstate analogue for thereaction catalyzed
by bakers yeast transketolase.This difference between transketolase and pyruvatedehydrogenase may
be related to differences in the polarity of the active
sites of the enzymes. It is conceivable that the active
site of the pyruvatedecarboxylase 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 transitionstate with no charge on the
thiazole portion of the coenzyme, similar to the uncharged 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 transition state. A charge-separated canonical form related
to TTPP could bepreferentially stabilized in the active
site of transketolase.

12406

Transition Stateof Transhetolase

DISCUSSION

The purpose of this study was to investigate the inhibition

of bakers' yeast transketolase by TTPP and the effect of
TTPP on 7-l for the activation of transketolase by thiaminPP. Fig. 2 shows the TTPP is not a tight binding inhibitor.
In this respect, transketolasediffers from the pyruvate dehydrogenase of E. coli (3) and bovine kidney (4). In those cases,
TTPP is a transitionstate analogue andinactivatesthe
enzyme almost irreversibly. Fig. 3 shows that TPPP is competitive with respect to thiamin-PP, and the Hill plots(Fig.
4) in the presence and absence of TTPP showed that TTPP
partially abolishes the negative cooperativity seen with thiamin-PP. The effect of TTPP on the cooperativity could be
due to the formation
of transketolase dimersin which TTPP
is bound to one subunit and thiamin-PP is bound to the
subunit. The negative cooperativity would bedecreased if
TTPP binding did not inhibit the binding of thiamin-PP to
the other subunit. Thiamin-PP binding to the first subunit
does cause such inhibition, as indicated
by the negative coopJ. C. Haggerty, 111, D. s. Shreve, and H. Z. Sable, (1983) Comput.
Biol. Med., submitted for publication.

erativity seen inFig. 3. This explanation assumes that transketolase exhibits true site-site interactions through
space and
that the cooperativityseen with transketolase is not a result
of the hysteresis (19).
Fig. 5 shows that T-' decreases with increasing concentrations of TTPP. This result was obtained whether reactions
were initiated with dimeror with a mixture of monomer and
dimer. The data of Egan and Sable (2) indicate that dimerization of inactive enzyme subunits may be one of the slow
steps in the activation of the enzyme by thiamin-PP. Other
possible slow steps could involve slow binding of thiamin-PP
or a slow conformational change in the enzyme, other than
dimerization. The decrease in7-l with increasing concentrations of TTPP could be due toa decrease in the rate constant
for some,as yetundefined, slow step in the activation
process.
Alternatively, the decrease in 7-l could be due to a decrease
in the concentrationof the enzyme form that undergoes the
slow step. The inhibitor could divert the enzyme into deadend,inhibitor-enzyme complexes that do not undergo the
slow conversion that is necessary for the activation. Studies
now in progress4 onthemechanism
of activation of the
enzyme by thiamin-PP may yield information that will help
us to understand the influence of these and other inhibitors
on 7-l.
Experiments in which dimeric apotransketolase was incubated either with or without TTPP and then assayed in the
presence or absence of TTPP (Table I) showed that TTPP is
not a slow binding inhibitor of transketolase. K, of TTPP
with transketolase is 28 8 nM in reactions initiated either
with dimeric apotransketolase or with mixtures of dimer and
monomer. This is nearly
two orders of magnitude greater than
the estimated upper limitof 0.5 nM for the Ki of TTPP with
pyruvate dehydrogenase (3). For oxythiamin-PP(II),a known
competitive inhibitor of transketolase, K , = 32 nM was found
in reactions initiated withdimeric apotran~ketolase.~
Gutowski and Lienhard,who first synthesized TTPP, considered it to be a transition state analogue resembling the
proposed
uncharged,
metastable
enamine
intermediate
formed during the decarboxylation of pyruvate by pyruvate
dehydrogenase or pyruvate decarboxylase (3). It seemed reasonable to postulate a related enamine intermediate as the
transition state during the transferof the keto1 group in the
transketolase reaction (1, 20). In the case of pyruvate dehydrogenase, the intermediate is the enamine form of the acarbanion of 2-(a-hydroxyethyl)thiamin-PP
(111);in the case
of transketolase, it should be the enamine form of the acarbanion of 2-(a,P-dihydroxyethyl)thiamin-PP(V). Despite
the superficial similarity between the enamine intermediates
in the two reactions, TTPP binds much less tightly to transketolase than to thedehydrogenase; Ki for TTPP (28 nM) is
anorder of magnitudelessthanthe
lowest K , for Mgthiamin-PP (0.4 p ~ (2).
) The replacementof the methylgroup
of 111 by an hydroxymethyl group in V results in a marked
increase in the polarity
of V relative to 111. AS a consequence,
TTPP is less appropriate as a transition state analogue for
the transketolase reaction than the pyruvate decarboxylase
reaction. This is reflected in its binding much less tightly to
transketolase than to pyruvate dehydrogenase. The differences in the affinity
of TTPP for transketolase and pyruvate
dehydrogenase must reflect considerable differences between
other
the active sites of the enzymes. Even though both require
thiamin-PP and both catalyze mechanistically similar reactions, they use differentsubstrates. Crosby et al. (21, 22)
reported that adductsof pyruvate with thiamin-PP are decar-

D. S . Shreve, J. C. Haggerty, 111, M. P. Holloway, and H. Z. Sable,

manuscript in preparation.

Downloaded from http://www.jbc.org/ by guest on September 21, 2014

phenomenonis observed both in the presence and in the

absence of T T P P , as shown in Fig. 3.
TTPP (160 nM) decreased the negative cooperativity seen
for thiamin-PP binding (Fig. 4). The slopes of Hill plots for
experiments inwhich TTPP was absent were 0.6 f 0.1 ( n =
2). This result agrees well with values previously obtained of
0.61 (2) and0.59.3The slopes of Hill plots for assay mixtures
containing 160 nM TTPP were 0.87 k 0.02 ( n = 4). So, for
thiamin-PP, in the absenceof TTPP, was 3.48 k 0.04 p~ ( n
= 2), in good agreement with a previously obtained value of 2
p ~ In . the~ presenceof 160 nM T T P P , So.5was higher, 6.8 k
0.9 p M ( n = 4).
Fig. 5 shows a plot of 7-l versus TTPP concentration for
an experiment in which the reaction was initiated with dimeric apoenzyme. At concentrations 2120 pg/ml, the apoenzyme is dimeric, but at lower concentrationsthere is an
equilibriumbetween monomeranddimer(14).Reactions
initiated with enzyme solutions containing40 or 50 pg/ml of
transketolase gave results similar to those shown in
Fig. 5, in
that 7-l decreased with increasingTTPP concentration. This
indicates that TTPP decreases the rate of activation when
the assay is initiated with dimeric apoenzyme as well as in
those initiated with a mixture of monomeric and dimeric
apoenzyme. Values of V, obtained in the experiment shown
in Fig. 5 were used to calculate K, for TTPP from a Dixon
plot (17) (Fig. 6). The data in Fig. 6 give a value of Ki= 26
nM. In other experiments in which 40 or 50 pg/ml of transketolase were used to initiate the assays, the Kiwas 37 and
22 nM, respectively.
Table I shows the effects on V8, and 7-l of incubation of
apotransketolase with Mg-TTPP prior to
assay. When dimericapoenzymewas incubated with Mg-TTPP and then
assayed in the presence of the same concentration of MgTTPP and 1 p~ thiamin-PP, the values of V,, and 7-l did
not differsignificantly from those observedwhendimeric
apotransketolase,incubatedinthe
absence of TTPP, was
assayed under the same conditions. Control studies, which
in
the assay mixture contained no TTPP and the enzyme had
not been incubated with TTPP, showed that, under the conditions used, TTPP inhibited the enzyme and caused a decreasein 7-l. The fact that 7-l is not decreased by prior
incubation of theenzymewithMg-TTPPindicatesthat
TTPP is not a slow binding inhibitor of transketolase (18).

Transition Stateof Transketolase

12407

REFERENCES
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. 2 5 6 , 48774883

22. Crosby, J., Stone, R., and Lienhard, G. E. (1970) J . Am. Chem.
SOC.92,2891-2900
23. Kremer, A.B., Egan, R. M., and Sable, H. Z. (1980) J. Biol.
Chem. 2 5 5 , 2405-2410
24. Kremer, A. B. (1981) Ph.D. Dissertation, Case Western Reserve
University, Cleveland, OH
25. Shreve, D. S., Holloway, M. P., Haggerty, J. C., 111, and Sable,
H. Z. (1983) Fed. Proc. 42,2178 (Abstr. 2458)

SUPPLEMENTARY MTERIAI

TO

D a v i d 8. Shreve. Uichilel P. H o l l o w d y ,
Henry 1. Sable

Jesse C. Haggerty. I l l and

L y o p h l l l z e db a k e r s 'y e a s tt r a n r k e t o l a r e
(E.C. 2.2.1.1) ( l o t 3lF-8025), l y o p h i l i z e d ag 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 phosphate ismerase (E.C. 5.3-1.11.
Rib-5-P , 11"-5-P and DPNH were o b t a i n e d f r o m Slgna. A l l o t h e r Chemicals were O f r e a g e n t

grade.
TABLE I
Effects an V,-

and 7 - l of P r e i n c u b a t i o n o f A p o t r a n r k e t o l a r e m t h

TTPP

Dimric apOtrdnlketollOe was I n c u b a t e d a t 25 'C for I h w r h 250

nn nPP, 3 n*l HgCI2 i n 42 rr*l T r i s - C l , pH 7.6. Controlincubations
i n c l u d e do n l y enzyme. wPCIz and T r i s - C l . Assays rere thenperfamed
i n t h e p r e s e n c e or absence O f 250 nM TTPP a s described ~n the Hethodl
s e c t i o n .A l l
valuerare presented d l average t S.D. VsI 1 5 ~n u n i t s
of AA$
,mni
and
i r i n u n i t so f .in-'.
The thiamin-PP
concentrat i o n = 1 VU. The preincubationmixturescontained
0.135 ng/d of
t r a n r k e t o l a r e4. r r a y r
w e ~ ei n i t i a t e dw i t h IO
opf 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 assays e r e Perfomred i n w a d r u D l i c a t e .
TTPP

I
"SI

In fimt ? n u b a t i o n
absent
Present

~n

absent

0.341

absent
p7esent

0.213

Present

0.20

0 009

0.56

0.07

0.007

0.31

0.05

0.27

0.04

t 0.01

Downloaded from http://www.jbc.org/ by guest on September 21, 2014

3. Gutowski, J. A., and Lienhard, G. E. (1976) J. Biol. Chem. 251,

boxylatedmorerapidly
as the polarity of the solvent de2863-2866
creases, and they hypothesized that the
active sites of all
4. Butler, J. R., Pettit, F. H., Davis, P. F., and Reed, L. J. (1977)
thiamin-PP requiring enzymes would be found to be hydroBiochem. Biophys. Res. Commun. 74, 1667-1674
phobic. In contrast, the active site of transketolase has been
5. Sykes, P., and Todd, A. R. (1951) J. Chem. Soc. Perkin Trans. I
found to contain charged amino
acids and is undoubtedly
534-544
6. Haggerty, J. C., 111 (1982) Ph.D.Dissertation, Case Western
more exposed to the aqueous solvent than is the
active site of
Reserve University, Cleveland, Ohio
pyruvate decarboxylase (23, 24).
7. Gutowski, J. A. (1979) Methods Enzymol. 62, 120-125
The results presented in this study suggest that no single
Berenblum, I., and Chain, E. (1938) Biochem. J. 3 2 , 295-298
structure can represent the transition states
of all enzymatic 8.
9. Gallo, A. A., Hansen, I. L., Sable, H. Z., and Swift, T. J. (1972)
reactions in which thiamin-PP is the coenzyme. The differJ. Biol. Chem. 2 4 7 , 5913-5920
ences among the transition states
will reflect at least the
10. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412
differences in structure and degree of polarity of the various 11. Ullrich, J., and Mannschreck, A. (1967) Eur. J. Biochem. 1,110116
active sites. The uncharged, enamine structure of I11 is appropriate for and could be stabilized by the apolar environ- 12. Heinrich, C. P., Noack, K., and Wiss, 0.(1972) Biochem. Biophys.
Res. Commun. 49,1427-1432
ment of the active siteof pyruvate decarboxylase. In contrast,
13. Belyaeva, R. Kh.,Chernyak, V. Ya., Magretova, N. N., and
interaction between the highly polar environment of the acKochetov, G. A. (1978) Biokhimiya (English Trunslution) 4 3 ,
tive site of transketolase (23, 24) and the uncharged, substi545-554
tuted thiazole portion of Va would be unfavorable. A more 14. Cavalieri, S. W., Neet, K. E., and Sable, H. Z. (1975) Arch.
Biochem. Biophys. 171, 527-532
appropriate formulationof this transition state is the chargeseparated canonical form Vb. Such a transition state would 15. Datta, A. G., and Racker, E. (1961) J. Biol. Chem. 236,624-628
be expected to be stabilized by complementary
charges in the 16. Neet, K. E., and Ainslie, G. R. (1980) Methods Enzymol. 64,
192-226
active site as well as bydiffusion of the formal,negative
17. Segel, I. H. (1975) Enzyme Kinetics, p. 110, Wiley-Interscience,
charge over the oxygen atoms of thea,P-dihydroxyethyl
New York
group.
18. Williams, J. W., and Morrison, J. F. (1979) Methods Enzymol.
63,437-467
Weconclude that TTPP is a competitive inhibitor for
bakers' yeast transketolase with respect to thiamin-PP but is 19. Ainslie, G.R., Jr., Shill, J. P., and Neet, K. E. (1972) J. Biol.
Chem. 247,7088-7096
not a slow, tight binding inhibitorof this enzyme. Because it
20. Krampitz, L. 0. (1969) Annu. Reu. Biochem. 78, 213-239
isnottightbinding,it
does notfunction effectively as a 21.
Crosby, J., and Lienhard, G. E. (1970) J. Am. Chem. SOC.92,
transition state analogue for transketolase.
5705-5716

12408

Transition Stateof Transketolase

R=

Downloaded from http://www.jbc.org/ by guest on September 21, 2014

TRANSKETOLASE (nM)

[TTPP]

t o a v a l u e on t h e a b r c i r r r

(nM)

r e p r e s e n t i n g -K,

(I71