Ribulose-1,5-diphosphate carboxylase and oxygenase from green plants are two different enzymes

BIOCHEMICAL

Vol. 81, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

March 30,1978

RIBULOSE-1,5-DIPHOSPHATE

539-546

CARBOXYLASE AND OXYGENASE FROM

GREEN PLANTS ARE TWO DIFFERENT ENZYMES

Rolf

Br;ind&n

Department of Biochemistry and Biophysics, University Gdteborg and Chalmers Institute of Technology, Fack, S-402 20 Gijteborg, Sweden Received

January

of

24,1978

SUMMARY: Ribulose-1,5-diphosphate oxygenase is shown to be an enzyme different from ribulose-1,5-diphosphate carboxylase. The enzymes can be separated from each other by a preparation method employing gel filtration at a pH of 8.3 which is higher is correlated than that previously used. The oxygenase activity with a blue color and a characteristic Cu EPR signal. This indicates that the oxygenase is a copper-containing enzyme.

An inhibition lower

of the

photorespiratory

rate

ductivity,

and several

inhibitors

(2).

carboxylase an oxygenase could

In

(EC

the

the

isolated

producing

activity

was sufficient

(4,5).

Therefore,

the

oxygenase

xylase *

activity

Abbreviations

it

1973

(2).

it

since used:

(3,4),

for

without

impairing

CO2 and 0

2

such

that

RuP2*

worked

as it

was responsible in vivo

and with

phosphoglycolate

photorespiratory

enzyme has attracted to achieve

pro-

although

contaminant

the

to a

plant

CO2 also

the amount of

seemed difficult function

fixing

Experiments

to account this

was reported

a minor

enzyme showed that

formed

may increase

phosphoglycolate that

leads

have been made to find

besides

4.1.1.39)

oxygenase

However,

and

of glycolate

This

(I).

attempts

1971

not be excluded

for

biosynthesis

rate

much attention an inhibition the

desired

were assumed to bind

(6). of

carboto the

RuP2 = D-ribose-1,5-diphosphate TRIS = Tris(hydroxymethyl)-aminomethan 0006-291x/78/0812~0539$01.00/0 539

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 8 1, No. 2, 1978

same site A new findings

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(7). approach in

this

and oxygenase

to

this

paper.

activities

enzymes.

Spectroscopic

contains

copper,

specific

inhibitors.

a result

problem It

is

is

shown

made possible that

the

by the

RuP2 carboxylase

are

associated

with

two

different

studies

indicate

that

the

oxygenase

the

search

that

might

help

in

for

MATERIALS AND METHODS: RuP2 carboxylase and oxygenase from parsley were prepared using TRIS-HCl buffer at pH 7.4 or 8.3, containing 2 mM fi-mercaptoethanol. Fresh parsley (200 g) and 400 ml of 50 mM buffer was turmixed in a Waring blendor at highest speed for 60 s. The suspension was filtered through 4 layers of cheese cloth and centrifuged for 10 min at 28 000 x g. Solid (NH412S04 was added to the supernatant, and the precipitate obtained at 30 - 50% saturation was dissolved in 50 ml of 5 mM buffer. The solution was applied to a Sephadex G-25 column (8 x 50 cm) equilibrated with the same buffer. The eluted protein fractions were concentrated in an Amicon ultrafiltration cell, supplied with a PM 30 filter. The concentrate (IO ml) was applied to a Sepharose 6B column (3.5 x 100 cm) equilibrated with the same buffer and eluted with a rate of 30 ml/h. Preparations were also performed in which 8-mercaptoethanol was omitted either in the last gel filtration step or in the whole procedure. In addition, RuP2 carboxylase and oxygenase were prepared from sweet pepper at pH 8.3 without B-mercaptoethanol. and glyceraldehyde-3RuP2, NADH, ATP, dithiothreitol -phosphate dehydrogenase were purchased from Sigma Chemical CO. Sephadex G-25 and Sepharose 6B were obtained from Pharmacia Fine Chemicals AB, Uppsala, Sweden. Phosphoglycerate kinase from bakers' yeast was prepared as described by Arvidsson et al. (9) and was a generous gift from T. Hjelmgren. All other chemicals were of analytic grade. Buffers were prepared from deionized distilled water. RuP2 oxygenase activity was determined at pH 8.3 from the oxygen consumption measured with a Clark electrode. The enzyme was activated as described by Lorimer et at. (IO), and 100 ~1 of the enzyme was added to 2.0 ml of 50 mM TRIS-HCl buffer, containing 20 mM MgC12. After about 10 min the rate of oxygen and 100 ~1 of 5 mM RuP2 was added. consumption was constant, The increase in the rate of oxygen consumption immediately after the addition was noted. RuP2 carboxylase activity was measured according to the method of Racker (II). Activated enzyme was added to 50 mM TRIS-HCl buffer at pH 8.3, containing 20 mM NaHC03, 20 mM MgC12, 1 mM NADH, 1 mM ATP, 1 mM dithiothreitol, 0.25 mM RuP2, phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase. Ascorbate oxidase activity was measured as described by Dawson et aZ. (12).

540

8lOCHEMlCAl AND 8lOPHYSlCAL RESEARCH COMMUNICATIONS

Vol. 81, No. 2,1978

RuP~CARSOXVLASE OXVGENASE ifi N

AND PEAK

-

2

FRACTION

NUMBER

Sepharose 6B gel filtration at pH 7.4 in the Fig. 1. preparation of RuP2 carboxylase and oxygenase from parsley. The buffer contained 2 mfi 8-mercaptoethanol. The absorbance at 280 nm is given in arbitrary units.

Total copper was determined with 2,2'-biquinoline (13). EPR spectra were recorded at 9.2 GHz and 77 K in a Varian E-3 and optical spectra were recorded in a Beckman spectrometer, Acta M IV spectrophotometer in a 1 cm cuvette.

RESULTS:

For

pattern

in

a preparation

the

last

gel

The RuP2 carboxylase same main if

the

peak

carboxylase shoulder If

agreement preparation

seen

fractions

in

and had the

the

was

spectrum

distributed

The EPR spectrum peak

from

of

a preparation

8-mercaptoethanol

in

consists

Fig.

2 were

shown

the

the

in

the

of a mixture

541

(4). the

1.

in

the

However, RuP2

and a distinct

preparation light

Fig.

entire

pH 7.4

Fig.

2). the

green-blue

3. At main

RuP2 carboxylase

made at

in

8.3,

(Fig. in

elution

appeared

separated

omitted

over

shown

to

peak

the

reports

raised

protein

shoulder

optical

earlier

was

was

is

activities

activities

on the

8-mercaptoethanol

at pH 7.4 step

with

and oxygenase was

same color

filtration

and oxygenase

in

pH of the

performed

pH 7.4 peak.

and oxygenase

in of

absence several

of signals

the

Vol. 81, No. 2, 1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 2. Separation of the RuP2 carboxylase and oxygenase activities from parsley on a Sepharose 6B column at pH 8.3. The buffer contained 2 mM 8-mercaptoethanol.

but in

is

mainly

the

(Fig.

due to

shoulder 4B)

obtained

which

is

the

same intensity

6.2

and If

pooled

added narrow other a half

of two

copper

and with

hyperfine

to

RuP2 resulted

shoulder

a Sepharose ratio

fractions

a cleaner signals

spectrum with

splitting

seen

in

6B column

between of the

dithiothreitol samples

hyperfine

time

the

about

constants

the

Fig. in

2 were absence

of

RuP2 oxygenase

copper

signal

seen

in

constant.

the

copper

the

intensity

ascorbate, to

composed

the

and the 4C is

If

However,

exhibit

in

8-mercaptoethanol

Fig.

at

4A).

pH 8.3

fractions

and applied

activity

(Fig.

mT, respectively.

13.3

the

copper

of

signal signal

of

in

Fig.

10

a very

4C)

min

at

slow

$-mercaptoethanol

3 and 4B the

disappeared

(Fig.

5 -

or

was

within reduced

color

was and the

30 s,

while

the

more

slowly

with

2S" C. The addition

of

1 mM

disappearance

542

of

the

color

8lOCHEMlCAL

Vol. 81, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 3. Optical spectrum of the green-blue fractions from parsley eluted from a Sepharose 6B column at pH 8.3 and having high RuP2 oxygenase activity. No $-mercaptoethanol was present in this preparation.

and the

narrow

The total

hyperfine

amount of copper

RuP2 carboxylase g-atoms given

in the

and oxygenase

extinction

coefficient

(4).

The EPR spectrum

of the total

pooled

activity

be 200,000

Sepharose

at

fractions

was about

0.2

based on the

280 nm for

in Fig.

the

having

previously

spinach

4B corresponds

to

20%

copper.

The molecular

weight

of the RuP2

- 300,000

from the

oxygenase

elution

was estimated

pattern

on the

6B column.

The shoulder constitutes removed

only.

per mole of RuP2 carboxylase,

protein

to

signal

in Fig.

more than

2 includes half

of the

by (NH412S04 precipitation

spectroscopic The relative

properties ascorbate

a contaminant protein without

and the oxidase

543

oxygenase activity

which

and which affecting

can be the

activity. of the

fractions

Vol. 81, No. 2, 1978

8tOCHEMtCAL AND BtOPHYStCAL RESEARCH COMMUNtCATtONS

I1

I 0.26

I 026

I

MAGNETIC

I1 0.30

FLUX

I 0.32

I

DENSITY

I 0.34

I

(T)

Fig. 4. EPR spectra at 77 K and 9.20 GHz of fractions eluted from a Sepharose 6B column in the preparation of RuP2 carboxylase and oxygenase from parsley. No B-mercaptoethanol was present in the preparation. (A) The main RuP2 carboxylase and oxygenase peak obtained at pH 7.4. (B) The green-blue fractions eluted at pH 8.3. (C) Same as (B) but frozen 30 s after the addition of 20 mM ascorbate. The modulat'on amplitude was 1 mT, and the gain settings were 1 x 10 5 3.2 x IO4 and 3.2 x 105 for (A) (B) and (Cl, respectively: The EPR parameters in (B) are fo; Type 1 Cu2+ = 6.2 mT and for the other type of copper = 13.3 mT.

with

high

copper ascorbate

RuP2 oxygenase

EPR spectrum

(Fig.

activity 4B)

(Fig. was

oxidase. 544

less

2) based than

3% of

on the that

of

BIOCHEMICAL AND BIOPHYSKAL RESEARCH COMMUNICATIONS

Vol. 81, No. 2, 1978

In the

preparations

green-blue

protein

spectrum

as that

DISCUSSION:

indications

pH caused

a decrease

oxygenase

activity

fraction These

splitting

in

the

Cu

1

easily

and the

from

2t

found

in

signal

two at

Fig. in

lower

while

with

2 in terms

the

between

increased

with

the

on reduction

the

"blue"

splitting

ref. of

narrow are

the 4).

two

hyperfine

both

most

1 Cu 2+ similar

Type

oxidases constant

to

The other

(14).

which

1 and 2 Cu 2+ in

of Types

is

inter-

the

"blue"

followed

the

RuP2

the

RuP2

(14). both

signals

and the

activity

which

oxygenase

contains

copper

the

amount

of

RuP2 carboxylase, considerable

oxygenase

of

The ratio

(see

a so called

oxygenase

For

(4).

be

several

activity,

explained

disappeared

those

However,

in

can

prepared

activities

now

were

protein

constant

are

which

presence

carboxylase

preparation

a hyperfine

to

oxidases

the

the

the

the

color

has

Although

there

for

EPR

RuP2 carboxylase

enzymes,

In retrospect,

of

a similar

parsley.

different

reports

same characteristic

with

evident'that

in

which

Type

mediate

two

Storing

originating

signal

is

the

enzymes.

The blue

the

2 it

from

and carboxylase

findings

likely

protein

remained

number

different

obtained

earlier

enzymes.

oxygenase

was

pH.

different

the

component

are

high in

pepper

Fig.

and RuP2 oxygenase at

sweet

of the

From

separated

from

future

strongly

the

amounts work

and more

it

firmly

that

many other is

oxygenase in

is

suggests

like

copper

color

the

small

oxygenases

compared

would

still

chloroplasts

necessary establish

545

to the

to

(8). that

be present

of green

purify amount

of

the

plants.

RuP2 and properties

BlOCHEMlCAL

Vol. 81, No. 2, 1978

of

its

Cu. The results

strongly

binding

do not disturb respiration

obtained

inhibitors

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

so far

to the

the RuP2 carboxylase

might

thus

be decreased

indicate

that

specific,

enzyme can be found activity.

The photo-

and plant

productivity

which

increased.

ACKNOWLEDGEMENTS: The author thanks Professor Carl-Ivar Brand& for manv fruitful discussions of this work. I also want to thank Professor Tore V&inngard for a valuable critical examination of the manuscript. This study was supported by grants from Statens naturvetenskapliga forskningsrzd.

REFERENCES: 1. 2.

3. 4. 5. 6.

Zelitch, I. (1975) Ann. Rev. Biochem. Vol. 44, pp. 123-145. Zelitch, I. (1975) CO2 Metabolism and Plant Productivity (Burris, R.H., and Black, C.C., eds.) pp. 343-358, University Park Press, Baltimore. Bowes, G., Ogren, W.L., and Hageman, R.H. (1971) Biochem. Biophys. Res. Commun. 45, 716-722. Andrews, T.J., Lorimer, G.H., and Tolbert, N.E. (1973) Biochemistry 12, 11-18. Andrews? T.J., Lorimer, G.H., and Tolbert, N.E. (1971) Biochemistry 10, 4777-4782. Laing, W.A., and Christeller, J.T. (1976) Biochem. J. 159, 563-570.

7. 8.

9. 10. 11. 12.

13.

14.

Chollet, R., and Anderson, L.L. (1976) Arch. Biochem. Biophys. 176, 344-351. Ullrich, V., and Duppel, W. (1975) The Enzymes (Bayer, P.D., ed.) Vol. XII B, pp. 253-297, Academic Press, Inc., New York. Arvidsson, L., Schierbeck, B., and Larsson-Raznikiewicz, M. (1976) Acta Chem. Stand. B 30, 228-234. Lorimer, G.H., Badger, M.R., and Andrews, T.J. (1976) Biochemistry 15, 529-536. Methods of Enzymatic Analysis (Bergmeyer, Racker, E. (1963) H ed. ) p. 188, Academic Press, Inc., New York. Methods of Enzymology D&on, C.R., and Magee, R.J. (1975) (Colowick, S.P., and Kaplan, N.O., eds.) Vol. II, Academic Press, Inc., New York. PP. 831-835, Brumby, P.E., and Massey, V. (1967) Methods of Enzymology (Estabrook, R.W., and Pullman, M.R., eds.) Vol. X, New York. PP. 463-474, Academic Press, Inc., Fee, J.A. (1975) Structure and Bonding Vol. 23, pp. I-60, Springer-Verlag, New York.

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