mecanismo de la transcetolasa bien

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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

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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

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and Ainslie, G. R.

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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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