BIOCHEMICAL

Vol. 71, No. 4, 1976

INHIBITION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF D-RIBULOSE

1,5-BISPHOSPHATE

CARBOXYLASE

BY PYRIDOXAL 5'-PHOSPHATE1 William

Whitman

and F. Robert

Department The University Austin, Received

June

Tabita

of Microbiology of Texas at Austin Texas 78712

16,1976

SUMMARY: Homogeneous D-ribulose 1,5-bisphosphate carboxylase from rubrum, Chlamydomonas reinhardtii, and Hydrogenomonas eutropha are low concentrations of pyridoxal 5'-phosphate. In the case of the Rhodospirillum rubrum, this inhibition is strongly antagonized by D-ribulose 1,5-bisphosphate. These results suggest that pyridoxal may act close to or at the ribulose 1,5-bisphosphate binding site from Rhodospirillum rubrum. The structural 1,5-bisphosphate EC 4.1.1.39)

properties carboxylase

Rhodospirillum

eucaryotes, subunits

and lacks

specific

activity

threitol

and EDTA (6).

which

subunits is Yet,

the --R. rubrum

For these

reasons,

model

system

(1).

retained

enzyme also

the

study

storage

respects,

of ribulose (dimerizing),

from

the photosynthetic

from other

containing

from higher

two large, this

the & plants.

from Rhodospirillum

procaryotes

rubrum Like

high

with enzyme

the higher

oxygenase rubrum

and

catalytic-type

enzyme exhibits

when reactivated

has a RuBP dependent

the RuBP carboxylase for

enzymes

Furthermore,

upon

in other

the enzyme

the

a dimer

subunit

carboxylase

with

Unlike

enzyme is

to the RuBP2 carboxylases

enzymes,

useful

small

be studied rubrum.

the &. rubrum

catalytic-type

(3-phospho-D-glycerate

may conveniently

bacterium,

similar

of the large

Rhodospirillum inhibited by enzyme from the substrate, 5'-phosphate of the enzyme

dithiois very plant

activity

(2,3).

may provide

of RuBP carboxylase.

'This work was supported by NSF grant BMS-7410297, a Biomedical Sciences Support Grant given to the University of Texas at Austin by NIH and by the University Research Institute of the University of Texas at Austin. 2Abbreviations used are : RuBP, D-ribulose 5'-phosphate. sulfonic acid; PLP, pyridoxal

Copyright 0 I976 by Academic Press, Inc. All rights of reproduction in any form reserved.

1034

1,5-bisphosphate;

MOPS, morpholinopropane

a

Vol. 71, No. 4, 1976

BIOCHEMICAL

PLP has been shown

to be a potent

phorylated

substrates

(4,5).

formation

at a lysyl

residue

hypothesis

with

the applicability

AND BIOPHYSICAL

inhibitor

of enzymes

The mode of inhibition at the

active

the RuBP carboxylase of these

RESEARCH COMMUNICATIONS

results

site.

is In this

from Rhodospirillum

usually

act

paper,

from

on phos-

by Schiff we test

rubrum

to RuBP carboxylases

MATERIALS

that

base this

and demonstrate

other

organisms.

AND METHODS

Rhodospirillum rubrum was grown on butyric acid as the electron donor and the RuBP carboxylase purified to electrophoretic homogeneity as previously described (6). -Protein concentration was determined from absorbance at 280 nm of 0.974 cm-l g-l 1 (1). Homogeneous Hydrogenomonas eutropha RuBP carboxylase was prepared by the procedure of Kuehn and McFadden (7). Protein concentration was determined by the absorbance at 280 nm of 1.55 cm-' g-l 1. Chlsmydomonas reinhardtii WT, strain 4, was grown and the RuBP carboxylase purified to homogeneity as described by Iwanij, et (8) except that the resuspended 55 per - al 2 cent saturated ammonium sulfate pellet was layered over a 0.1 - 0.3 M continuous sucrose gradient and centrifuged for 20 hours in the SW 25.1 rotor at 23,000 RPM at 0-4'~. The protein concentration was measured by the biuret method with crystalline bovine serum albumin as a standard (9). The absorption at 280 nm in 10 mM potassium phosphate, pH 7.5, and 1 mM EDTA was then determined to be 1.45 cm-' was isolated from spinach leaves obtained at the g-l 1. Spinach RuBP carboxylase local market by the procedure of Paulsen and Lane (10). The barium salt of RuBP was prepared by the enzymatic procedures described except that in the recrystallization step, equivalent by Horecker, et al. (ll), The barium salt amounts of acetic acid were substituted for hydrochloric acid. was converted to the free acid with Dowex H+ resin. RuBP obtained by this procedure was 80 to 90 percent pure with the major contaminants being inorganic phosThis preparation was chloride free. phate and presumably ribulose 5-phosphate. Commercial preparations of RuBP were never as pure as the enzymatically generated substrate. Prior to treatment with PLP, enzymes were incubated for 3 hours at 30°C in the presence of 10 mM dithiothreitol, 1 mM EDTA, and 10 mM MOPS, pH 7.5. This activated enzyme was dialyzed for at least 3 hours against a 500 fold or greater Stock solutions of PLP were volume of the same buffer without dithiothreitol. prepared prior to each experiment and the concentration determined by the absorbance at 388 nm of 6050 cm-1 M-l in O.lN NaOH. Except where noted, incubations of the enzymes with PLP were for 60 minutes at 3O'C. The incubation was terminated by the addition of the enzyme to the assay solution containing PLP at the same 0.8 mM RuBP, 20 mM [14C]NaHC03, 20 mM Mg acetate, concentration as in the incubation, After 10 minutes, 0.1 1 mM EDTA, and 0.15 M MOPS-KOH, pH 7.8 in a 0.25 ml volume to ml of 99 per cent propionic acid was added and the free [ 14 C]C02 allowed 14C remaining was determined by counting dissipate. The amount of acid stable 0.2 ml in 3 ml of liquid scintillation cocktail containing 930 ml toluene, 70 ml and 4 g of 2,5-diphenyloxazole per liter in a Beckman Biosolv BBS-3 (Beckman), LS-1OOC liquid scintillation counter. RESULTS A comparison of buffers buffers

of PLP inhibition

at pH 7.5 lacking

(Fig.

inorganic

1) showed anions.

of the &. rubrum that

inhibition

In the presence

1035

RuBP carboxylase was most

pronounced

of MOPS-NaOH,

greater

in a variety in than

Vol. 71, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

loor-

’

’

-MOPS-NsOH

0.2

0.3

[PLP]

Fig.

1.

Comparison of PLP*inhibition buffers. 9 x lo-" M 3. minutes in the presence bis tris propane-HCl, or the absence of PLP. Per the presence of PLP/CPM

P L

2.

50 per

0.4

(mM)

of &. rubrum RuBP carboxylase in different was incubated for 120 --rubrum RuBP carboxylase and absence of PLP in 0.033 M potassium phosphate, MOPS-NaOH at pH 7.5. Assays were performed in cent activity remaining is expressed as (CPM in in the absence of PLP) x 100.

-

Hydrogenomonos

-

Rhodospiriflum

euiropho rubrum

60

l

b > = 4 I=

Fig.

q

40 20 0

0.2

0.1

0.3

0.4

Titration of RuBP carboxylase from three microorganisms with PLP. Incubations and assays were performed as described in Materials and Methods. The specific activity in micromoles CO2 fixed/min/mg of the enzymes in the absence of PLP were: lJ. rubrum, 4.9; C. reinhardtii, 0.35;H. eutropha, 0.60.

cent

potassium

phosphate

was obtained enzyme with phosphate, remained KOH could

inhibition

with

was obtained and bis

34 and 33 per absence

be substituted

cent

mM PLP. greater

In a similar

experiment,

0.1 mM PLP, and either activity

of either for

0.02

propane-HCl

0.1 mM PLP.

MOPS-NaOH buffer,

in the

tris

with

remained

anion.

MOPS-NaOH with

1036

It

while

In the presence than

50 per after

cent

inhibition

incubation

of the

10 mM NaCl or 10 mM Na only

was subsequently no effect

of

17 per

cent

found

on the inhibition

that

activity MDPSby PLP.

Vol. 71, No. 4, 1976

BIOCHEMICAL

When RuBP carboxylase Hydrogenomonas

eutropha

inhibition

at low

titrated

in a similar

at low that

Table

I:

activity

concentrations

with

fashion,

the 5.

rubrum

of PLP.

In data

not

activity

Per

cent

of activity

1,5-bisphosphate

81

ribulose

5-phosphate

47

5-phosphate

37

inorganic

34

To determine

phosphate

was 0.05 mM. of RuBP used was 0.8 mM; the other controls

without

more about

structurally

PLP in the presence

PLP remained assays

as compared were

PLP but

the site

enzyme was treated

3) and other

Furthermore,

acid

34

aPLP concentration bThe concentration at 1.6 m&i. 'Calculated from present.

without

remainingC

45

6-phosphogluconate

with

found

RuBP carboxylase

ribulose

3-phosphoglyceric

bation

was also

32

ribose

(Fig.

it

by PLP.

none

the &. rubrum

enzymes were

was more inhibited

here,

of I& rubrum

Protectorb

substantial

these

RuBP carboxylase presented

and

2),

While

was inhibited

of PLPa inhibition

reinhardtii

PLP (Fig.

of PLP was evident.

RuBP carboxylase

Protection

RESEARCH COMMUNICATIONS

of the Chlamydomonas

enzymes was titrated

concentrations

spinach

AND BIOPHYSICAL

linear

with

each potential

of PLP interaction

with

PLP in the

related

to 37 per at least

1037

cent

(Table cent in

tested compound

RuBP carboxylase,

of the substrate I).

of the

After control

the absence

6 minutes

were protector

with

presence

compounds

of RuBP, 82 per

for

compounds

after

RuBP

a preincuactivity

of RuBP. preincubation

in

Vol. 71, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TIME OF ASSAY

Fig.

3.

Protection of PLP inhibition of 2. rubrum RuBP carboxylase by RuBP. 4 x lo-' M R. rubrum RuBP carboxylase was incubated in the presence and absence of 0.05 mM PLP, 0.8 mM RuBP, 1 mM EDTA, and 3.5 mM MOPS-KOH, pH 7.5 in a 0.06 ml volume. After 60 minutes, 0.05 ml was withdrawn and added to the complete assay mixture in a 0.75 ml volume. At 2 minute intervals, 0.1 ml of the assay mixture was added to y22 ml of propionic acid in a liquid scintillation vial and acid stable C measured as described in Materials and Methods.

the presence

or absence

observed

longer

for

tration.

Other

to the extent phosphate

of PLP and RuBP (Fig.

assay

could

times

phosphorylated of RuBP.

The small

cannot

The failure

account

amount

is

consistent

their

from

by lowering to protect

that

and low with

Deviations

of protection

the extent

of 6-phosphogluconate

to protect

failed

indicate for

3).

be abolished

compounds

and ribulose-5-phosphate

preparations

acid

(MIN.1

the enzyme concen-

against

observed

PLP inhibition with

these

contaminants

of protection

seen with

concentrations lack

linearity

inorganic of the RuBP RuBP (Table

I).

of 3-phosphoglyceric

of effect

on enzyme activity

(6,12).

DISCUSSION Pyridoxal

5'-phosphate

RuBP, structurally inhibition. suggests Since from s.

related

However, that

allosterism rubrum

both

PLP may act

is

a potent

inhibitor

compounds,

and inorganic

the

and specificity

close

extent

the

locus

anions

with

of PLP interaction

1038

RuBP carboxylase.

protect

of protection

to or at a RuBP binding

has not been demonstrated (1,6),

of I& rubrum

respect

site

of the

against

this

by RuBP enzyme.

to RuBP for

may be at the active

the

enzyme site

of

BIOCHEMICAL

Vol. 71, No. 4, 1976

this

protein.

inhibited lation

The fact by low

is

a highly

the

existence

for

the spinach

with

the

discuss

throughout specific

aspect

RESEARCH COMMUNICATIONS

diverse if

sources

the mechanism

is

also

of carboxy-

evolution. reagent

for

lysyl

RuBP carboxylase of lysyl

from

of PLP is expected

of two essential

importance this

RuBP carboxylase

concentrations

conserved

PLP is

that

AND BIOPHYSICAL

(13).

residues.

of PLP action

lysyl

residues

residues per

in proteins.

large

subunit

The results

reported

A subsequent

report,

in greater

depth

Recently,

has been proposed here

are

consistent

in preparation,

will

(14).

ACKNOWLEDGMENT The authors and Jerry Brand

wish to thank Roy Repaske for for his assistance in culturing

providing the Hydrogenomonas Chlamydomonas.

cells

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Tabita, F.R., and McFadden, B.A. (1974) J. Biol. Chem., 249, 3459-3464. McFadden, B.A. (1974) Biochem. Biophys, R.&. Commun., 60, 312-317. N.E. (1974) Biochem. Biophys. Res. Ryan, F.J., Jolly, S.O., and Tolbert, Commun., 59, 1233-1241. of Proteins, Means, G.E., and Feeney, R.E. (1971) Chemical Modification Holden-Day, Inc., San Francisco. p. 134. Colombo, G., and Marcus, F. (1974) Biochemistry, l3, 3085-3091. Tabita, F.R., and McFadden, B.A. (1974) J. Biol. Chem., 3, 3453-3458. Kuehn, G.D., and McFadden, B.A. (1969) Biochemistry, 8, 2394-2402. Iwanij, V., Chua, N.-H., and Siekevitz, P. (1974) Biochem. Biophys. Acta, 358, 329-340. and David, M.M. (1949) J. Biol. Chem., Gornall, A.G., Bardawill, C.J., 117, 751-766. Paulsen, J.M., and Lane, M.D. (1966) Biochemistry, 2. 2350-2357. A. (1958) Biochem. Prep., 5, 83-90. Horecker, B.L., Hurwita, J., Weissback, Tabita, F.R., and McFadden, B.A. (1972) Biochem. Biophys. Res. Commun., 48, 1153-1159. Norton, I.L., Welch, M.H., Hartman, F.C. (1975) J. Biol. Chem., 250, 80628068. Whitman, W., and Tabita, F.R. Manuscript in preparation,

1039