The binding of thiamine pyrophosphate with transketolase in equilibrium conditions

Vol. 63, No. 4,1975

THE BlNDING

BIOCHEMICAL

OF THlAMINE

AND BIOPHYSICAL

PYHOPXOSPBATE WlTH TBANSKBTOLASE IN

BQUIIJBXLUM G.A. Laboratory

Kochetov,

N.K.

CONDlTlONS

Tikhomirova,

Pebruary

10,

P.P.

Philippov

Lomonosov USSR

of Bioorganic Chemistry, Moscow 117234,

Received

RESEARCH COMMUNICATIONS

State

University

,

1975

SUkMAHY: The binding between thiamine pyrophosphate and transketolase, purified from baker’s yeast, in e uilibrium conditions has been studied. In the presence of Ca 2!+, the enzyme molecule has been shown to possess two binding site! for the coenz whose dissociation constants are 3.2 x IF and 2.5 x IO- Fe’ M; besides, there are site(s) where the binding of the coeneyme is cooperative inIn the presence of Mg2+, a positive less firm. teraction between the binding sites of thiamine pyrophosphate has been observed. Regardless of the cation used, the major part of the catalytic activity of the transketolase molecule manifests itself in the binding of one molecule of the coenzyme.

In ketolase mine

1961

it

was shown by Hacker

(!!K)

molecule

pyrophosphate

which

the

died

(2-101,

but

TPP binding

2.

In

in

the

on Sephadex,

TK purified

of Ca2+ and Mg2’ quantity Copyright AN rights

on there

the

the present baker’s

under

the

o 19 75 by Academic Press, Inc. of reproduction in any form resewed.

were

(6,g)

yeast equilibrium

paper

in were

that

the

stu-

number

was equal the

removed

to

presence by gel

of filt-

of protein-bound the

binding

was studied

in

conditions,

TPP was determined 924

in

quantity

thia-

some works

and it

then

a transof

analogues

was preincubated

which

from

of protein-bound

two works

cofactors

after ln

were

‘TPP and its

in

that

(I)

2 to 9 molecules

TK was determined,

enzyme

free

TPP was determined. with

Later

was only

sites

the

from

of TK with it

cases

TPP and Mg 2+, ration

(TPP).

interaction

of

both

may bind

and Datta

in

of ‘TPP the i.e.

the

presence the

presence

Vol. 63, No. 4,1975

BIOCHEMICAL

of an equilibrium

AND BIOPHYSICAL

concentration

RESEARCH COMMUNICATIONS

of the coenzyme.

METHODS TK, after al.

having been purified

was fractionated

(II),

lution

by the method of Racker et

twice or thrice

of ammonium sulphate

(pH 7.6);

with a saturated

the resulting

so-

preparations

of the enzyme were homogenous, as proved by the data of polgacrylamide

gel electrophoresis.

parations tivity

was

10

measured directly,

determined

25'C in both cases). by the optical

nm (the optical

density

density value

activity

of the pre-

or 20 U if

measurements TK was preincubated

(temperature

14.5

U if

The specific

prior

with 'I'PP and metal ion

The quantity

of protein

value of '!!!I(solutions

of 'I% TK solution

(12) ) or by the method of Lowry (13). of the measurements were multiplied

The TK activity

(14)

previously.

as described

in (15)

(1.4

The binding

with

phosphate buffer the required

(pH 7.6),

concentrations

containing

3.6p, tion 3.5

by a factor

were determined

columns

with 2.5 mM potassium1.0

mM CsC12 or MgC12 and 25'C).

was varied

in the presence of A6g 2+- from 0.2 to 3.6p; of gel filtration

of 0.7.

of TPP with TK was studied

of TPP (temperature

presence of Ca2+, the TPP concentration

case

and the re-

the help of Sephadex G-50

x 28 cm), which were equilibrated

at 280

In the latter

and the quanti tg of TPP (I>

as described

was

at 280 nm is

bovine serium albumin was used as standard protein sults

to ac-

In the

from 0.02 the dura-

was 2.5 hours in the former case and

hours in the latter.

The results on TPP-TK binding were treated in r/A vs r coordinates (161, where r is the value of the ratio of the number of protein-bound

TPP to the number of moles of TK (molecular

925

BIOCHEMICAL

Vol. 63, No. 4, 1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

2 . : 1 .. . 2 In i

:ig.

3indi ng of ‘I’PP wi til 2K in 62g2+ (B). For experimental Straight lines in Yig. IA res method.

1.

weight

140,000 (1) >, A is the concentration

our case the concentration to its

total

sing a different is bound

most

M, the site

there are several affinity

firmly

value

cases. It in Fig.

binding

is not excluded, IA is indicative

sites

zyme binding perimental In this

curves

is unknown,

than in the first

cooperative

two

interaction

An approximate

between the coen-

extrapolation

gives an r

the experiments 926

from that in the

of the r/A vs r dependence interaction

to the abscissa

case, unlike

constant

different

curve

cooperative (17).

x low8

between TPP and TK in the pre-

IB) is basically

sites

K'=3.2

(17).

of the binding

the positive

posses-

however, that the dependence shown

case of Ca2+. IJ!he bell-shaped reflects

sites

= 2.5 x low7 M; in addi-

much higher

of negative

between the TPP binding sence of Mg2+ (Fig.

constant

- I("

that

where the coenzyme

whose dissociation

is undoubtedly

The character

as indicating

independent

has a dissociation

of less firm

equal

obtained in the presence

to TPP. The site

there is a site(s)

but its

The results

IA) should be interpreted

in the ‘I’K molecule

of free TPP (in

of free coenzyme is practically

concentration).

of Ca2+ (Rig.

tion,

the pre:;\+nce of Ca 2+ (A) and conditions see “kiie tbods”. are drawn by the least squc;-

values

of the exclose

with Ca2+, no direct

to

2.

deter-

Vol. 63, No. 4, 1975

0.08

ifig.

;i.

BIOCHEMICAL

AND BIOPHYSICAL

t

Dependence of ,i’i; activitg on ‘22F aresence ol Ca2+ (A) and L1g2+ (8). termined cfter Gel filtration in rcted by a buffer containin&; Y!?P details see “fLethocis”). StraiLnt least squares method.

mination

of

studying

the dependence

the

concentration

is

KD value

(Fig. u,

possible,

of

2B).

t

t 60

V

--0---

/

0

this

. l

16 .0

can be done

activity

(v)

constant

(Kz)

by

on TPP deter-

.

.

0 l

l 0* 0

I I

0

I

0

I

1

3.

but

The dissociation

%

80

concentration in the ‘I’ii activity was deSephadex G-50 e uiliband the cation ‘zfor lines are drawn by the

transketolase

100

r'it;.

RESEARCH COMMUNICATIONS

0

I

2 3 z Jkpcndencc of LX activity on the amount of proteinbound ,i’i?P. Reconstruction of holo-TK was carried out in the presence of Ca2+(0) or I,$+ (0); ( - - - ) theoretical graph for the case v:hen the enzyme mol.ecule contains only one DC tive center and vaiue K for active i’i?l? molecule is much lower ihan for nonactive one(s). Activity measured in the presence of the optimal conand ‘PW was kken for IO@;. centra tions of the cation

927

Vol.63,No.4,1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mined in this way equals 3.0 x the case of Ca2+ (Pig. to 7.4 x

in which the binding

the major part,

Ca2+,

if

not all,

of the TK molecule is revealed in the binding

the TH activity obtained

the at every

3 that in the presenof the catalytic

in the binding

acti-

of one TPP

of the other molecule of the coenzyme,

insignificantly. Although the results in the presence of Mg2+ are not so unambiguous, it is

nevertheless vity

of TPP with

we also measured the ,I'K activity

r . It is seen in Pig.

given value of

molecule;

in

10-s.

apoenzyme was studied,

vity

calculation

2A) gives for TPP a value of KE equal

In the experimenta

ce of

M; a similar

10m7

increases

evident

that here too the major part of the acti-

should be referred

to the first

of the two bound TPP mole-

cules. DILSCUSSION It vity

has been demonstrated

of TK reveals

itself

that

the maximum catalytic

when approximately

come bound to a mole of the enzyme (6,g). on reconstruction

and the quantity

If

acti-

2 moles of TPP beno metal is added

of bound TPP is measured af-

ter the free coenzyme has been removed, the enzyme activity increases

linearly

as r changes from 0 to 2. In the presence

of Mg2+, the linearity

is not observed so strictly,

over 60% of the maximal TK activity if

was revealed

the removal of free TPP is carried

for a longer at least

time,

the percentage

7Q%regardless

the data of this

clusions: site

+l) either

(6). However,

out on larger

columns and

of the TIC activity

at ~1

of whether the metal (calcium

magnesium) is added or not (our unpublished plus

and at J.Y

data).

and/or

These data

study prompts one to the following

in the TK molecule

and the other TPP binding

site(s) 928

is

con-

there is only one active performs

a different

BIOCHEMICAL

Vol. 63, No. 4, 1975

function;

2)

sistes

to

the

the

of ‘TPP bound

different

the

with

certainty sites,

cooperative

or in

the

sites

possessing

is

not

lated.

the

pectively. from referred

to the

of

centre If

the

is

when

drawing

the

valid,

which

rison

is

3.0

x 10-7

in

possible M is

weakly

true,

then

fact

favour for known.

the of

the

the But,

second

to be bound, are

mutually

may be calcu-

M, res-

of Ca 2+ determined x low8

M and should site

if

the

two active

hypothe-

sites

speaking, K;#K;‘).

taken

is

the

will

that

in

as only the

be extent

No such

Mg2+,

this

account

to a certain

with

of

But if

into

hypothesis.

remembering

be

the Kh and Kg values

Ki
experiments

929

interconver-

x 10m7

(i.e.

This

case

ki and 2.5

strictly

unequality

(The

the pre-

and not

case.

coenzyme.

YPP in

about

expressed

sites,

for

i.e.

than,

a ne-

TPP binding

ligand

binding

be nonlinear

graph, in

7.4

valid,

either

sites

the presence is

is

constants

x low8

suggestion

should

nonlinearity

an ar$ument

is

is

curve

is

in

between

possibly

obtained

“stronger”,

effectivity

vs l/A

thus 3.2

the

TPP binding

dependence

first,

coincide.

different

to

value

one active

should

l/v

equal

vs l/A

l/v

values

of

of Bng2+ one may

to the

of dissociation

presence

independent

several,

affinity

be paid

the ‘!WP binding

to

values

the

cooperativity

affinities

the

in

case

several

in

and

should

Ca2+ there

with

If

The Ki

the

the

here).

of Ca 2+ are

binding

positive

are

different

The Ki and Ki’

sence

sis

there

may exists

discussed

the TK activity

between

different

with

independent,

the

whereas

TK molecule

forms

In

interaction

when an enzyme tin@;,

of TPP-TK

about

two active

attention

enzyme,

IA and IB).

are

effiCienCieS.

between

the

character

TPP binding

ga tive

to

RESEARCH COMMUNICATIONS

there

catalytic

relationship

Ca2+ and Mg 2+ (Fig. speak

the TK molecule

different

from

amount

in

else

possessing Apart

the

or

AND BIOPHYSICAL

cornpaKg =

experiments

Vol. 63, No. 4,1975

sith

BIOCHEMICAL

Ca2+ the values

of q

AND BIOPHYSICAL

and l$ differ

RESEARCH COMMUNICATIONS

two-fold

at the most,

the l$ value for TPP in the presence of BQ2+ may be estimated frosn the corresponding

I$.

The authors thanks T.Xhaifsts tion

of this

for

assistance

in prepara-

paper. REFERENCES

Datta, A.G. and Racker, E. (1961) Kochetov, G.A., Usmanov, R.A. and Letters 2, 265-266. G.A. and Usmanov, R.A. 3. Kochetov, Commun. 4l, 1134-1140. G.A., Izotova, A.E. and 4. Kochetov, Biochem.Eiophys.Res.Commun. 43, G.A. and Izotova, A.E. 5. Kochetov, ::

J.Biol.Chem. 236, Merzlov, V.P.7970) (1970)

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Kochetov, G.A., va, N.K. (1972) C.P., 7i Heinrich, Eur.J.Biochem. G.A. a. Kochetov, 6.

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Heinrich, C.P. (1973) Experientia 2, 1227-1228. Heinrich, C.P., Schmidt, D. and Noack, K. (1974) Eur.J.Biothem, 41, 555-561, Tabachnick, 1~1. and Racker, E. Srere, KA., Cooper, J.R., (1958) Arch.Eiochem.Biophys. 74, 295-305. Heinrich, C.P., Noack, K. and Wiss, 0. (1972) Biochem.Biophys. Res.Commun. 49, 1427-1432. Lowry, O.H., Rosenrough, N.J., Farr, A.L. and Randall, R.J. R&l;,5;) J.Biol.Chem. 193, 265-275. E. (1961) in:?Ih-e Enzymes, vol. 5, pp. 397-406, Acade&c Press, New York and London. Hummel, J.P. and Dreyer, W.J. (1962) Biochim.Biophys.Acta

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