Interaction of ribulose diphosphate carboxylase with 2-carboxyribitol diphosphate, an analogue of the proposed carboxylated intermediate in the CO2 fixation reaction

BIOCHEMICAL

Vol. 48, No. 3, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INTERACTION OF RIBULOSE DIPHOSPHATE CAPBOXYLASE WITH 2-CARBOXYRIBITOL DIPHOSPHATE, AN ANALOGUE OF THE PROPOSEDCARBOXYLATED INTERMEDIATE IN THE CO2 FIXATION REACTION. Marvin I. Siegel and M. Daniel Lane Department of Physiological Chemistry The Johns Hopkins University School of Medicine Baltimore, Maryland Received

June 19,1972

SUMMARY

Support for the participation of a six-carbon carboxylated intermediate (or transition state) in the D-ribulose-1,5 diphosphate carboxylase-catalyzed reaction was derived from kinetic and binding studies with 2-carboxy-D-ribitol-1,5-diphosphate (CRDP), an analog of the proposed intermediate and potent inhibitor of the reaction. Evidence is presented that following the formation of a carboxylase.Mg*CRDP complex, a slow temperature-dependent conformational change occurs locking the enzyme in an inhibited state from which release of the inhibitor is extremely slow.

Earlier

investigations

in this

2-carboxy-D-ribitol-1,5-diphosphate potent

inhibitor

of

the

laboratory

(1) have

(CRDP, Structure

ribulose

diphosphate

I),

shown that is

a

carboxylase-catalyzed

reaction. CH2-OPO;I C(OH)-CO; I HCOH I HCOH

CH -OPO;I * C(OH)-CO; I c=o I HCOH

I

I

CH2-OPO;-

CH2-OPO;-

I. It

was suggested

its

structural

II. (1) that

similarity

CRDP acts

as an inhibitor

to the hypothetical

mediate,

2-carboxy-3-keto-D-ribitol-1,5-diphosphate

proposed

by Calvin

the enzymatic pated

that

the

(2) or a closely-related

reaction. inhibitor

Copyright 0 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.

Were this would bind 508

the

because

carboxylated

inter-

(Structure transition

case,

of

it

more tightly

state

II) in

would be anticithan

either

sub-

BIOCHEMICAL

Vol. 48, No. 3, 1972

strate,

i.e.

ribulose

structural

of both.

intermediate

consistent

with

The present

other

site

or CO2, since

The participation

or transition findings

communication

the active

state

provides

evidence

characteristics

carboxylated

six-carbon

it

contains

of

either

the

in the reaction

on the enzymatic

and has the

the hypothetical tion

diphosphate

elements

proposed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

mechanism

that

(3).

CRDP acts

at

of an analogue

of

intermediate

or transi-

state.

METHODS Ribulose units

diphosphate

per mg)

carboxylase

was prepared

(specific

activity,

in homogeneous form

1.2-1.5

from spinach

leaves

and assayed by the methods of Paulsen and Lane (4). Unlabeled and 14 [carboxyC] CRDP were chemically synthesized and chromatographically-purified

as described

showed that (41,

is

binds

earlier

the carboxylase

composed of

8 moles

of enzyme

8 pairs

(1).

Previous

investigations

has a molecular

weight

of nonidentical

subunits

of D-ribulose-1,5-diphosphate

of

5.6 x lo5 (5,6)

and

or CRDP per molecule

(1).

RESULTS Exposure to a lo-fold fold

excess

sites

of ribulose

diphosphate

stoichiometric

excess

over

the

per molecule

enzymatic

activity

CRDP/enzyme ratios, endpoint

(1)

(see Fig.

1).

the

binds

The presence in the

extent

incubation

This

and the

with

magnesium ion, reaction, of enzyme with

509

20-40 min

enzyme

(a 1.25-

causes almost

binding

complete

loss

that

at lower

fact

achieved

to CRDP concentration

carboxylase-catalyzed

the preliminary

),

for

substrate

of inhibition

stoichiometrically

of divalent

of CRDP over

diphosphate

of enzyme

is proportional

inhibitor

(4)

8 ribulose

carboxylase

respect

at

the

suggested

that

to active

an essential is

of

time

the

sites.

activator

also

required

CRDP for

maximal

during inhibi-

BIOCHEMICAL

Vol. 48, No. 3, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

[CRDP] /

1

/-

bl

O/I

x,.-x

3/l

7/l

IO/l

-Ye--

IO PRELIMINARY

20 INCUBATION.

MINUTES

Fig. 1. Dependence of the loss of carboxylase activity on CPDP Homogeneous ribulose concentration during preliminary incubation. diphosphate carboxylase (1.8 x lo-8M) was incubated at 30° in a reaction mixture containing 0.2 M T is-Cl buffer, pH 7.8, 10 mM M [14C] KHC03 (1.2 x 10 5 c.p.m. per vmole), 0.06 mM MgC12, 0.05 EDTA, and 6 mM g#utathione in thy absence or presence of 5.4 x 10m8M, 1.3 x lo- M, or 1.8 x lo- M CPDP. After preliminary incubation for varying periods of time, 0.9 ml aliquots were withdrawn, mixed with 0.1 ml of 7 mM ribulose diphosphate, and the rate of ribulose diphosphate carboxylation determined during a 10 min assay.

tion ing

(Fig

2).

On the

substrate

species

CRDP (results

not

other

hand,

(7),

is not

our earlier

observation

(1) that

carboxylase

is

only

ions,

eg.,

Mg2+ or Mn2+,

equimolar lated

amounts of

by gel It

is

centrations ditions, dependent

tight

for

inhibitor

the

inhibition

are consistent binding

in the presence

and that

the carboxylat-

of

and divalent

with

CPDP to the

of divalent

complexes

by

formed

metal

metal contain

ion when iso-

filtration.

interesting

slow process

dioxide,

required

These results

shown).

observed

carbon

requiring of

inhibitor

not only

the

that

inhibition

by CRDP is a relatively

20-40 min to reach employed extent,

but

upon CPDP concentration.

510

(see Fig. also

the

Direct

completion

Under these

1). rate

at the

of inhibition

binding

experiments

conconis

BIOCHEMICAL

Vol. 48, No. 3, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Additions h4gz* CRDP “One

CRDP

I

I 5 PRELIMINARY

I IO

I 15 INCUBATION,

+ hQ2+

I 20 MINUTES

I

Fi Dependence of inhibition by CPDP upon the presence of ** The preliminary incubation preliminary incubation. Ms9.+ during and carboxylase assays were carried out as described in Fig, 1, The but with the variable additions indicated in the Figure. concentrations of ribulos diphosphate carboxylase and CPDP were 1.8 x 10m8M and 1.8 x lo- 7 M, respectively.

1 “0

6

; t

2

4

6

8

IO

MINUTES

Fig. 3. Kinetics of ribulose diphosphate carboxylation after incubation varying periods of preliminary ;ht;he CRDP. Preliminary incubation of 1.8 x 10 -8; ~~;;~=;;;~~ absence (Control) or presence of 1.3 x 10B7M CKDP was carried out as described in Fig. 1; at 1,2,5,10,20 and 40 min, aliquots were taken for carboxylase assays as in Fig. 1.

511

BIOCHEMICAL

Vol. 48, No. 3, 1972

-eo

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CRDP

I

PRELIMINARY

INCUBATION,

MINUTES

Protection of the carboxylase by ribulose diphosphate Fig. 4. from inhibition by CRDP. Preliminary incubation of the carboxylase (1.8 x 10 -8M) was conducted as described in Fig. 1 except for the variable additions indicated (0.7 mM D-ribulose diphosphate and 1.8 x 10m7M CFLDP) and the omission of [lk] KHCO3. After varying periods of preliminary incubation, aliquots were withdrawn and carboxylase assays initiated by the addition of [14C] KHCO3 (final concentration, 0.05 M) and ribulose diphosphate (only when not present in the preliminary reaction mixture).

(1) show that time

dependence.

ditions

drawn for

in Fig.

occur

at all.

yielded

the presence diphosphate

since

CRDP and ribulose site,

the presence the

Fig. phate

of

or

site ribulose with

Furthermore, CRDP with

release

during

the

but

linear,

This

supported

carboxylase

512

conslow

3, enzyme withincubation

during

produces

for

substrate of evidence.

preliminary

prevents of

significant

to compete

diphosphate

CRDP effectively interaction

extremely

is particularly

by two lines

diphosphate

the

either

the

carboxylation rates -4 (7 x 10 M) of rib-

appear

ribulose

a similar

enzyme under

in Fig.

concentration

assay.

the is

is

exhibits

the preliminary

diphosphate

apparently

carboxylase

4).

carboxylase

As illustrated

a high

in the

A common binding

of

its

reduced, of

ulose

binding

1,

assay at any point

inhibitor

despite

to the

Once CRDP is bound to the

outlined

or does not

with

CRDP binding

either

qualitatively

site. First,

incubation

inhibition ribulose

a common

(see diphosidentical

BIOCHEMICAL

Vol. 48, No. 3, 1972

difference both

spectra

spectra

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the ultraviolet

exhibit

sharp

spikes

296 nm and 285 nm, and a broad The height ribulose

of

the

diphosphate

inhibition

reaches

of

10, the

lytic

activity Despite

at low the

I), ally

(4O),

of

assay'.

Merely

absence of

the

inhibited

I,

ribulose

I),

is

by titrating

temeratures complex of

cata-

(see Table

which

is catalytic-

the complex

fully

to the

complex,

active

diphosphate,

promotes

activity.

Enz + Mg2' + CRDP -

spectrum

to the carboxylase

to 30° for

(Experiments

by CBDP can be resolved

of

from difference

the complex

in the presence

on catalytic

dissociation

7-8 moles of CRDP bound per mole of

a temperature-dependent

carboxylase

nearly

288 nm peak

the carboxylase-inhibitor

Table

warming

state

(30")

upon exposure

at 4" and having

the

tightly

an enzyme*Mg.CPDP only

produces

l), the

of CRDP/

1).

CRDP binds at higher

ratio

determined

(Fig.

Hence,

(Experiment

effect

as,

occurs

the

to that

that

as well

enzyme

(Fig.

265 run.

to a maximum with

which

calculated

inhibitor

temerature.

prepared

through

identical

fact

formation

carboxylase

complex,

with

inactive

higher

is

the

a condition

5,

as minima at

maximum at approximately

The KD (- 10S8M) for

CPDP from the ternary data,

at 288 nm, as well

or CBDP; at a concentration

a maximum.

titration

As shown in Fig.

288 nm peak can be titrated

enzyme of approximately total

region.

II

a brief its

period

transition

A and B, Table

I),

in to

presumably

conformation

change. Warming the 2+ or absence of Mg has no detectable Thus,

kinetically 4oY

in the standard

it into

appears

two steps

EnzsMg '+.CRDP _i) 300

1

that

inhibition (shown below).

(EnzmMg2+.cRDp)'

In direct kinetic experiments as in Fig. 1, preliminary incubation of the enzyme with CEDP and Mg2+, but at 0°, results in no inhibition in the subsequent assay at 30°.

513

BIOCHEMICAL

Vol. 48, No. 3, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Factors

affecting

I

inhibition

by and binding

of

CRDP

In Experiment I, 14.8 mg of ribulose diphosphate carboxylase were incubated at 4" for 3 hours in 1 ml containing 0.1 M TrissCl buffer, pH 8 at 4', 0.5 mM [carboxy-14C] CRDP (1.5 x 10' d.p.m. per umole), and 2 mM MgS04. The mixture was applied to a 0.9 x 41 cm column of Sephadex G-75 and eluted at 4O with 0.1 M Tris+Cl, pH 8.0, containing 0.1 mM EDTA. After pooling the carboxylasecontaining fractions, protein concentration, carbox lase activity, and CRDP bound were determined. Controls without [ Y4C] CRDP in the preliminary incubation mixture were processed in an identical manner. In Experiments II, III, and IV, gel-filtered carboxylase from Experiment I containing 7-8 moles of CRDP per mole of enzyme was subjected to preliminary incubation at 30° with 0.2M TriseCl buffer, pH 7.8, containing either: 1) Experiment II - 50 m&l KHC03, 10 mM MgC12, 6 mM GSH, and 0.1 mM EDTA; 2) Experiment III50 InM KHCO 0.7 mM ribulose diphosphate, 1.0 mM EDTA and 6 mM GSH; or 3T'Experiment IV - 0.5% sodium dodecylsulfate (SDS) 0.1 mM EDTA, and 5 m&l 2-mercaptoethanol. Following preliminary incubation, the reaction mixtures were gel-filtered and the eluates analyzed as in Experiment I except that in Experiment IV the eluting buffer also contained 0.5% SDS.

Preliminary incubation conditions

Experiment

Enzyme after gel filtration moles mole relative speCRDP/ enzyme cific activity* % 7-8 94

180 min at 4" with[14C] CRDP + Mg2+

I. II.

III.

IV.

gel-filtered enz from I; 10 min at 30° with Mg2+ 30 min at 30° with Mg2+

7 7

16 11

gel filtered enz from I; 10 min at 30° with RuDP f 1 mM EDTA

2

91

gel-filtered 10 min at SDS "activity manner,

enz from I; 30° with 0.5%

0

to those of CRDP omitted

controls treated in Experiment I.

relative but with

At 4O an enzyme*Mg*CRDP ternary reversed,

whereas,

further,

but

hibited

state.

at 30° this

irreversible Unlike

complex ternary

change which ribulose

is

diphosphate

514

formed which

complex locks

in an identical can be

can undergo

a

the enzyme in an inand CO2 which

have

BIOCHEMICAL

Vol. 48, No. 3, 1972

I 270

I 280

I 290

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 1 300 310 WAVELENGTH,

I 320 nm

I 330

I 340

Difference spectra of ribulose diphosphate carboxylase Fig. 5. promoted by D-ribulose-1,5-diphosphate and CRDP. The difference spectrum (lcm light path, 3 ml total volume) of 3 mg of homogeneous carboxylase in 0.25 M TrisaCl buffer, pH 7.8, 0.1 mM EDTA, 5 mM 2-mercaptoethanol and 1 mM D-ribulose-1, 5-diphosphate versus the same mixture minus ribulose diphosphate was recorded The effect of at 30“ with a Cary model 15 spectrophotometer. Mg2+ in the presence of ribulose diphosphate could not be tested, since the presence of traces of HCO?J, which contaminate the reaction mixture despite the use of "C02-free" reagents, allows the enzymatic reaction to proceed. The difference spectrum promoted by 1 mM CRDP was determined as described above except that 10 mM MgC12 was present in both the experimental and reference cuvets. easy access of both

substrates

be expected site.

state, The fact

active

once this

"tightening" with

the

CPDP which

greater

difficulty

kinetic

barrier

of

inhibitor,

would markedly that

site,

and has an additional

to encounter

However,

formational contacts

to the

the

site

perhaps

strengthen

its

the Mg2+ -dependent

515

has structural

negative

charge

gaining is

binding

binding

would

access

surmounted,

to accomodate as occurs

elements

all

to the

a conof

its

in the transition and prevent

of CPDP to the

release. carboxylase

BIOCHEMICAL

Vol. 48, No. 3, 1972

under

these

(Fig.

5) supports

the

formation

iments the

conditions

III

occurs total

view

the

and IV,

carboxylase

induces

the

of

since incubation

release

with

that

its

that

complex.

removal

It

is

is

bound

effected

accompanies clear

to

activity

loss

diphosphate with

(Exper-

covalently

without

EDTA and ribulose

inactivation

change

change

CRDP is not

partial

with

spectral

a conformational ternary

I)

RESEARCH COMMUNICATIONS

a characteristic

inactive

Table

upon

AND BIOPHYSICAL

sodium

and its dodecyl

sulfate. The results Calvin's phate

proposal intermediate

catalyzed

reaction.

intermediate

is

presented (2) that

in

this

II)

Work on the

ACKNOWLEDGEMENTS These investigations National Institutes of

strongly

support

a 2-carboxy-3-keto-D-ribitol-1,5-diphos-

(Structure

currently

communication

in

is

involved

chemical

progress

in

in

the

synthesis our

carboxylaseof

the

proposed

laboratory.

were supported by a grant Health, USPHS (AM-14575).

from

the

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Wishnick, J. Biol. Calvin, Siegel, 3rd ed., Academic Paulsen, Rutner, 28, 531 Rutner, Cooper, J. Biol.

M., Lane, M.D., and Scrutton, M.C., Chem., 245, 4939 (1970). M., Federation Proc., 13, 697 (1954). M.I., Wishnick, M., and Lane, M.D., in The Enzymes, Ed. by Boyer, P.D., Vol. 6, pp. 169-192, Press, New York (1972). J.M., and Lane, M. D., Biochemistry, 2, 2350 (1966). A.C., and Lane, M.D., Biochem. Bionhvs. Res. COmmunS., (1967). Biophys. Res. Communs., 2, 923 (1970). A., Biochem. T.G., Filmer, D., Wishnick, M., and Lane, M.D., Chem., 244, 1081 (1969).

516