Vol. 88, No. 1, 1979

8lOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 14, 1979

Pages 85-91

MODIFICATION

OF RIBULOSE BISPHOSPHATE

CARBOXYLASE FROM RHODOSPIRILLUM RUBRUM WITH TETRANITROMETHANE'

Peter

D. Robison

Department The University Austin, Received

March

and F. Robert

Tabita'

of Microbiology of Texas at Austin Texas 78712

12,1979

SUMMARY: Ribulose bisphosphate carboxylase from Rhodospirillum rubrum is inactivated by low concentrations of tetranitromethane. A$$ition of the substrate ribulose 1,5-bisphosphate and preincubation with Mg and HCO - both protect against inactivation. A spectrum of modified enzyme shows a3peak at 430 nm, consistent with modification of tyrosine residues. This modified enzyme contains 1.07 nitrotyrosine residues under conditions where 49% of the activity is lost. These results provide the first evidence for an essential tyrosine residue at the active site of ribulose bisphosphate carboxylase.

INTRODUCTION Ribulose fer

1,5-bisphosphate

of CO2 to RuBP3 and is

phate

or Calvin

cycle

(1).

carboxylase the initial

rubrum

is a convenient

because

simple

quaternary

is

a dimer

(2),

of large

the higher

contains

8 large

Recent cysteine

(6)

1 Supported Foundation 2Author

plant

subunits

chemical

tool

structure.

physical Whereas

and has a native

and 8 small

modification

subunits

studies

and arginine(8,9,22)residues

weight

of about of unknown

have implicated for

requests

should

studies enzyme

of 114,000 560,000

(3)

and

function.

lysine

by National Institutes of Health Grant GM-24497, Grant F-691, and by NIH Postdoctoral Fellowship

to whom reprint

microorganism

the &. rubrum

as essential

phos-

and chemical

molecular weight

the trans-

pentose

the photosynthetic for

enzyme has a molecular

(catalytic)

catalyzes

step .in the reductive

The enzyme from

Rhodospirillum of its

(EC 4.1.1.39)

(4-7),

activity.

Prelim-

Robert A. Welch GM-07817 to PDR.

be sent.

3 Abbreviations

used are: RuBP, ribulose 1,5-bisphosphate; (hydroxymethyl) aminomethane; DEAE, Diethylaminoethyl; dinitrilo) tetraacetic acid.

Tris, tris EDTA, (ethylene-

0006-291X/79/090085-07$01.00/0 85

Copyrighr AN rights

@ 1979 by Academic Press, Inc. of reproduction in anyform reserved.

BIOCHEMICAL

Vol. 88, No. 1, 1979

inary pyl)

investigations

in our

-carbodiimide

be present methane,

a mild

In this nitrating

on RuBP carboxylase

shown

from pea and spinach

(12).

using

indicated

study

1-ethyl-3an essential

we have examined

reagent

that

to cross-link

(3-dimethylaminoprotyrosine

the effect

selectively Higher

from &. rubrum.

have been previously

MATERIALS

laboratory

hydrochloride

(10).

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

modifies

levels

subunits

might

also

tetranitro-

tyrosines

(ll),

of tetranitromethane of the eucaryotic

enzyme

AND METHODS

RuBP carboxylase from &. rubrum was purified as in previous procedures (13) except for the elimination of the streptomycin step and the addition of a Bio Gel A 1.5 M gel filtration step after chromatography with DEAE cellulose. Sodium dodecyl sulfate gel electrophoresis (14) showed the Protein concentrations of the enfyme were calculatenzyme to be >95% pure. ed from the extinction coefficient at I&O nm of 0.974 Lg (2). Enzyme activity was measured by acid stable [ C] NaHC03 incorporation (15). The assay mixture (0.25 mls) contained 40 mM Tris-SO (pH=8.0),10 mM dithio0.8 mM RuBP and 20 mM NaH t!O3 containing 2 uCi of ;~t;;;t;;kc;O mu W12, 2 VZi of [ 14c1 . The assays run with 1 mM NaHCO also contained NaHCO . Ths enzyme was activated by incubatian with all assay components excep 2 RuBP for 5 minutes. The assay was then initiated with RuBP and terminated after 5 minutes by the addition of propionic acid. IncuTetranitromethane solutions were made up fresh in 95% ethanol. bations with tetranitromethane were performed in 50 mM Tris-SO4 (pHz8.0) Concentrations of up to 10% at room temperature unless otherwise indicated. The reaction was ethanol in incubations had no effect on enzyme activity. terminated by the addition of 25 mM dithiothreitol or 70 mM 2-mercaptoethanol. The spectrum of nitrated enzyme was run on a Cary 219 spectrophotometer operFor spectral analysis, modified and control ating with an automatic baseline. (incubated in the presence of the same amount of ethanol minus the tetranitromethane) enzyme were dialyzed overnight against 50 mM Tris-SO , pH=9.0. TO control and modif 4 ed enzyme from determine total protein sulfhydryl content, the spectral analysis were subsequently dialyzed overnight against 0.08 M sodium phosphate buffer (pH=8.0) containing 2% sodium dodecyl sulfate and content was then measured as in Habeeb (16). 0.5% EDTA. The sulfhydryl RuBP was prepared enzymatically from ribose 5-phosphate (17), as described earlier (15). Tetranitromethane ~8s from Aldrich or Sigma; samples from both sources behaved identically. [ C] NaHC03 was from Amersham/Searle. All other materials were of reagent grade. RESULTS Incubation

of &. rubrum

tetranitromethane (Fig. activity about

1).

caused

A molar within

8%of

RuBP carboxylase

a rapid,

low

concentrations

concentration-dependent

excess

of 17 fold

caused

one hour.

A higher

excess,

the activity.

with

The substrate

86

a loss

loss of about

35 fold,

RuBP afforded

caused

of

in activity

70% of the the loss

significant

pro-

of

Vol. 88, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

I

I

1

IO

20

30

40

50

60

Time

Fig.

Inactivation of R. rubrum RuBP carboxylase with tetranitromethane. The control activity Each incubation xor~m 9.7 uM enzyme. per mg protein. 0, 84 uM tetranitrowas 1.51 umol HCO fixed/min , 336 pM tetranitromethane. methane; A, 168 pi tetranitromethane;l

1.

tection

against

Using

the loss

17 fold

the loss

excess

from

against

loss

were

The effect centrations I,

incubated

inhibits

afforded

Since tyrosine

1).

and NaHC03 protected

of 2.5 mM Mg 5 minutes

+2

and 20 mM

before

reduced

lowering

with

at low

the activity

tetra-

the loss

in

against

to the higher

level,

+2 2.5 mM Mg .

Fructose

tetranitromethane of the carboxylase is

thought

to react

the pH to 6 eliminates

87

(1 mM) NaHC03 con-

using

at 20 mM NaHC03 and activates

the product

tetranitromethane

on activity than

In contrast

no protection

(19),

the enzyme for

2).

of 0.4 mM RuBP reduced

+2

maximum activation,

greater

activity

phosphoglycerate,

with

Mg

The addition

of tetranitromethane

by 1 mM NaHC03 when present which

the addition

(Fig.

50%.

was slightly Expt.

2).

to ensure

by tetranitromethane

In addition,

35%. (Fig.

addition to about

caused

tetranitromethane,

of activity

nitromethane activity

of activity

70% to about

NaHC03, which

(Table

(min)

20 mM NaHC03

there

1,6 bisphosphate, at 1 mM NaHC03 (10,18),

inactivation reaction with almost

was no protection

as did (Table

the phenoxide all

I,

3Expt. ion

of the nitration

2). of

Vol. 88, No. 1, 1979

BIOCHEMICAL

‘O. 1

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 20

IO IO

, 30 Time

Fig.

I 40

I 50

I 60

(min)

2. Protection from tetranitromethane inactivation of RuBP Carboxylase by substrates. Each incubation contained 9.7 uM+snzyme and 168 pM tetranitromethane. A, 0.4 mM RuBP; 0, 2.5 mM Mg and 20 mM NaHC03; 1, no additions.

Table

I. Effect Tetranitromethane

of Low NaHC03 Inactivation

Activity, Effectorsaand of RuBP Carboxylase % Activity

Expt.

pH on

Remaining

Additions Standard Assay 20 mM NaHC03 10 11 33

IlOne

+2 1 mM NaHCO + 2.5 mM Mg 20 mM NaHCa3 + 2.5 mM Mg'2

1 mM RuBP 1 mM Fructose 1,6bisphosphat 1 mM 3-phosphoglycerate

17 63 15 12

pH=8.0 pH=6.0 pH=6.0

9 67 44

*0lle

pH=6.0 aIncubations were tetranitromethane. maleate.

UOIE IlOllS

20 mM+pHC03 a-i Mg 0.5 mM RuBP for

+ 2.5

1 mM NaHC03

Assay

3 7 31

71

1 hour and contained The pH=6.0 incubations

88

9.7 uM enzyme were performed

and 168 uM in 50 mM Tris-

Vol. 88, No. 1, 1979

of this

BIOCHEMICAL

residue.

With

by tetranitromethane at the higher

pH (Table

I,

with

is

which

are

nitrotyrosine

inactivation

duced

inactivation

sines

per enzyme molecule

This

that

bicarbonate

(10)

seen

enhanced i-2 - Mg

carboxylase

acid)

is

and indicates

tetranitromethane

caused

nitration

3).

of tyrosine

control

probably

occur-

Using

nitrotyrosines

per

an extinction

of both

control

groups,

no sulfhydryl

were

groups

activity.

present

would

in

a broad

A peak

for

was calculated modification

nitration

protein

pro-

of two tyro-

be sufficient

enzyme showed

this

oxidized

it This

A total

and modified

were

(20),

indicating

subunit)

(11).

coefficient

enzyme molecule.

(one per

spectral

residues

enzyme showed

-1 cm-1 at pH=9.0

RuBP carboxylase

sulfhydryl

.

was also

the -___ R. rubrum

of 49% of the activity

of all

+2

than

is

versus

430 nm (Fig.

were

that

with

of 4200 mol liter 1.07

less

by tetranitromethane

enzyme with

of modified

there

2.7

groups

consistent

spectrum

determination

This

was far

(bis-2-nitrobenzoic

of the

at about

ination

3).

this

some inactivation

pH.

Modification

centered

but

seen when

of sulfhydryl

at the lower

difference

also

5,5'-dithio

some oxidation

changes

Expt.

RESEARCH COMMUNICATIONS

from &. rubrum,

of 20 mM NaHC03 and 2.5 mM Mg

inactivation

incubated

ing

RuBP carboxylase

was seen at pH=6,

by the addition enhanced

AND BIOPHYSICAL

for

elim-

sulfhydryl the same amount,

enzyme preparation,

by tetranitromethane

indicating during

the in-

cubation. DISCUSSION Incubation

of RuBP carboxylase

tetranitromethane ylating

activity.

phosphoglycerate this

or the

effector

the loss one tyrosine

of approximately

with

low amounts

and concentration-dependent

of the substrate,

Preincubation

against

of approximately

a rapid

Addition

inactivation.

protected

loss

caused

from -*R ~rubrum

fructose

RuBP, but not 1,6 bisphosphate,

per

50% of the activity

89

enzyme dimer indicates

the product protected

by tetranitromethane.

residue

of carbox3against

NaHC03 and Mg+2 also

of the enzyme with

of activity

loss

of

that

The nitration with

a subsequent

a stoichiometry

of

Vol. 88, No. 1, 1979

8lOCHEMlCAL

0.06

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-

I

I

I

I

340

360

300

400

1

I 420

I 440

Wavelength

Fig.

is

sufficient

to the stoichiometry modification

carboxylase

NaHC03 stimulates phosphate

(nm)

results

by nitration

subunit.

per dimer

activity than

inactivation.

5'-phosphate

residue

rather

total

(21).

is

inactivation is

sufficient

Moreover,

protects

This

where for

the addition

the inactivation

in contrast the

loss

of all

of Mg+2 and

caused

by pyridoxal

(4).

These vated

for

of pyridoxal

of 1 lysine

&. rubrum

further

I 480

3. Difference spectrum of tetranitromethane-modified versus control RuBP carboxylase. The modified enzyme (10.7 pM) was incubated with 168 PM tetranitromethane for 1 hour and treated as in Materials and Methods.

one per subunit

acid

I 460

Thus,

at the active characterize

indicate

that

RuBP carboxylase

of one specific a nucleophilic site this

tyrosine

group,

tyrosine,

of RuBP carboxylase. tyrosine

modification.

90

from&.

rubrum

of the 12 present

is

inacti-

(2)

on each

may be an important

amino

Work is

in progress

to

Vol. 88, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. .9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

28, 379-400. Jenson, R.G., and Bahr, J.T. (1977) Ann. Rev. Plant Physiol. B.A. (1974) J. Biol. Chem. 249, 3459-3464. Tabita, F.R., and McFadden, Paulsen, J.M., and Lane, M.D. (1966) Biochemistry 2, 2350-2357. Whitman, W.B., and Tabita, F.R. (1978) Biochemistry 17, 1282-1287. Paech, C., and Tolbert, N.E. (1978) J. Biol. Chem. 253, 7864-7873. Schloss, J.V., Stinger, C.D., and Hartman, F.C. (1978) J. Biol. Chem. 253, 5707-5711. Chollet, R., and Anderson, L.L. (1978) Biochim. Biophys. Acta 525, 455467. Chollet, R. (1978) Biochem. Biophys. Res. Commun. 2, 1267-1274. Lawlis, V.B., and McFadden, B.A. (1978) Biochem. Biophys. Res. Commun. so, 580-585. Whitman, W.B. (1978) Ph.D. Dissertation, University of Texas at Austin. Riordan, J.F., and Vallee, B.L. (1972) in Methods in Enzymology, Vol 25, pp 515-521, Academic Press, New York. Grebanier, A.E., Champagne, D., and Roy, H. (1978) Biochemistry 17, 51505155. Tabita, F.R., and McFadden, B.A. (1974) J. Biol. Chem. 2, 3453-3458. Laemmli, U.K. (1970) Nature 227, 680-685. Whitman, W., and Tabita, F.R. (1976) Biochem. Biophys. Res. Commun. 71, 1034-1039. Habeeb, A.F.S.A. (1972) in Methods in Enzymology, Vol 25, pp 457-464, Academic Press, New York. Horecker, B.L., Hurwitz, J., and Weissback, A. (1958) Biochem. Prep. 5, 83-90. Whitman, W.B., and Tabita, F.R. (1978) Fed. Proc. 2, 1426. Bruice, T.C., Gregory, J.J., and Walters, S.L. (1968) J. Amer. Chem. Sot. 90, 1612-1619. Sokolovsky, M., Riordan, J.F., and Vallee, B.L. (1966) Biochemistry 2, 3582-3589. Whitman, W.B., and Tabita, F.R. (1978) Biochemistry 17, 1288-1293. Schloss, J.V., Norton, I.L., Stringer, C.D., and Hartman, F.C. (1978) Biochemistry 17, 5626-5631.

91