Modification of histidine at the active site of spinach ribulose bisphosphate carboxylase

Vol.

94, No.

June

30.

BIOCHEMICAL

4, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 1091-1097

1980

MODIFICATION

OF HISTIDINE AT THE ACTIVE SITE OF SPINACH RIBULOSE BISPHOSPHATE CARBOXYLASE

Ashok

K. Saluja

and Bruce

A. McFadden

Biochemistry/Biophysics Program Washington State University Pullman, WA 99164

Received

March

31,198O

SUMMARY: Ribulose 1,5-bisphosphate carboxylase from spinach was rapidly inactivated by diethylpyrocarbonate (DEP) at pH 7.0 and 30°C. The inactivation showed saturation kinetics with a half-inactivation time at saturating DEP equal to 0.1 minutes and KDEP = 7.4 mY. One substrate, ribulose bisphosphate, the product 3-phosphoglycerate and two competitive inhibitors protected against inactivation, thereby indicating that DEP modifies the active site. DEP-modified enzyme showed an increased absorption at 240 nm which was lost upon treatment with 0.4 M hydroxylamine. Most of the activity lost by DEP modification could be restored after treatment with 0.4 M hydroxylamine at 4°C. The results suggest that DEP modified 2 to 3 histidine residues per 70,000-dalton combination of large and small subunits. These residues are essential to catalysis by the carboxylase activity of ribulose bisphosphate carboxylase/oxygenase. INTRODUCTION: glycerate Calvin

D-ribulose-1,5-bisphosphate

carboxylase cycle

RuBP* with

(dimerizing)

and from most, CO2 to give

is (2)

that

it

is

known about and Whitman

at the active site

the

site

(8)

the criteria

catalyzes

of 3-phosphoglycerate

of its (3),

for

(4-6)

has suggested

and plant

the modification to establish

in the

site. the

The presence (5)

of the

(l),

presence

of a lysine

of arginine

of sulfhydryl

groups

1091

in spite nature

of these

of

O2 to give

by Hartman

has also

active site are yet to be met. * RuBP = D-ribulose-1,5-bisphosphate; Abbreviation: (also known as ethoxyformic anhydride).

the reaction

Research

enzymes

the placement

of the

or with

However, protein

active

of the carboxylase.

of the bacterial

but

structure

[3-phospho-D-

a key catalyst

autotrophs,

the most abundant

and Tabita

evidence

not all

is

and one phosphoglycolate.

probably

is also

if

EC 4.1.1.391

two molecules

one 3-phosphoglycerate fact

carboxylase/oxygenase

little et al. residue

at the active

been shown. (7) residues

There

and tyrosine at the

DEP = diethylpyrocarbonate

DOO6-291X/80/121091-07$01.00/0 Copyright 0 I980 by Academic Press, Inc. All rights of reproduciion in any form reserved.

Vol.

94, No.

4, 1980

BIOCHEMICAL

Diethylpyrocarbonate pH values,

reviewed Since

in aqueous

has been shown

considerable

specificity (10).

(9)

residue

histidine

and the for

BIOPHYSICAL

solutions

to modify

The pH optimum

a histidine

AND

at neutral residues

use of this

as a base

COMMUNICATIONS

or slightly in proteins

reagent

RuBP carboxylase

may function

RESEARCH

acidic with

has been recently

activity

is

about

8.0.

the

2,3-

in deprotonating L

enediol

of RuBP (ll),

examined

in the

the effect

present

of histidine

modification

by DEP" has been

work.

MATERIALS AND METHODS: DEP, 3-phospho-D-glyceric acid, N-acetylcysteine and tetrasodium salts of RuBP and sedoheptulose-1,7-bisphosphate were obtained from Sigma Chemical Co. Carboxyhexitol-1,6-bisphosphate was prepared by the method of Gordon et al. (12). NaHl4C03 was purchased from ICN. Other chemicals used during this Evestigation were of a reagent quality. To obtain the enzyme for this investigation, the commercial preparation from spinach (Spinacia olenacea) was obtained from Sigma Chemical Co. and further purified by sedimenting 3 mg/ml of the crude enzyme into a 0.2-0.8 M linear sucrose gradient prepared in a TEMB buffer containing 50 ml4 Tris, 1 mM EDTA, 20 mM MgC12.6 H20 and 50 mM NaHCO (pH 8.0 at 25°C). The final specific activity of the enzyme which was homoge 2 eous by the criterion of polyacrylamide gel electrophoresis was 1.5 pmole CO2 fixed/min/mg protein. For modification studies the enzyme was concentrated (1-2 mq/ml) by dialysis in vacua against TEM (pH 7.0 at 25°C). The enzyme was incubated with the indicated concentration of DEP (dissolved in ethanol) at 30°C. After the stated time, an aliquot of the incubation mixture was added to a quenching buffer (TEM containing 20 mM histidine, 20 mM NaHCO and 2 mM dithiothreitol, The enzyme treated in the absence of prot 2 cting pH 8.0). agents was assayed as described below. In studies of protection an aliquot of the incubation mixture was diluted with 19 volumes of quenching buffer before assay. For the assay of RuBP carboxylase (13) the reaction mixture at pH 8.0 (25°C) contained in 250 U1: 2.4 umoles MgC12, 0.4 umole EDTA, 0.1 ,mole dithiothreitol, 6 umoles NaHl4CO (spec. act. 0.2 uCi/pmole), 5 Pmole Tris-Cl, 0.25 umole RuBP, 2.0 pmole histl 4. ine and lo-40 Pg of the enzyme. The reaction mixture (except RuBP which was contained in 50 ~1) was preincubated for at least 10 min at 30°C before initiating the reaction at 30°C with RuBP. In the case of rotection experiments the reaction was initiated by a mixture of RuBP and NaH'; 4CO3. The reaction was terminated with 60% cold trichloroacetic portion was transferred to acid 2 min after the addition of RuBP. An aliquot a scintillation vial and excess 14C02 was liberated at room temperature overThe activity was measured as RuBPnight prior to radioactive counting. dependent incorporation of l4CO2 into acid-stable product. RESULTS: with

RuBP carboxylase

low concentrations

inactivated vation presence

when

appears

from

rapidly

lost

of DEP at pH 7.0 at 30°C.

incubated

with

to be pseudo-first

of 1 mM DEP.

spinach

The time

1 mM DEP at 30°C for order course

with

activity

when

For example,

it

10 minutes.

a tl,2

of 1.3 minutes

of inactivation

at different

1092

incubated was completely The inactiin the DEP

Vol.

94, No.

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

80

IA 0 L

IC

0

2

I

[Cont.

0

I

2

TIME Figure

1.

T =-t

3 mM]-’

3

4

OF INACTIVATION

5

(Min.)

Inactivation of spinach RuBP carboxylase by DEP at 30°C. o-o, Enzyme (E) + ethanol (control);v --v, + 0.32 mM DEP;n--0, + 0.65 mM DEP; e-o, + 1.0 mM DEP; x-x, + 1.3 mM DEP; and A-A, + 2.6 mM DEP. DEP, in ethanol, was freshly prepared before each experiment and the concentration of DEP was determined spectrophotometrically at 240 nm after reacting with at least a lOOO-fold excess of histidine. The inset shows a plot of half-inactivation time, T, versus l/[OEP].

concentrations In

of DEP

is

2

shown

y$&

in

Fig.

(14),

1.

The

where

7 is

"pre-equilibrium" the

model

half-inactivation

predicts

time.

that

Thus

a

k2 plot

of

minimum at

vs.

1

a saturating

with

T . minimum

saturation active

should

m half-inactivation concentration = 0.1

prior

a straight

time,

as to

i.e., of

minute

kinetics site

give

and

would

modification,

be

the

DEP. KDEp

line time

The

mM. if

presumably

1093

y

intercept

required

insert

= 7.3

expected

with

in Thus

DEP was at

the

for Fig. the

equal

the

half-inactivation 1 shows

such

inactivation

reversibly same

to

shows binding

site.

a plot

the

Vol.

94, No.

4, 1980

BIOCHEMICAL

Table

1:

Protection against

AND

BIOPHYSICAL

of Spinach Inactivation

RESEARCH

RuBP Carboxylase by DEP

Percent after Concentration of Organic Phosphate

Additions

Noneb

---

DEPC

_-_

10 Min

(100)

(100)

14

0

89

62

DEP + sedoheptulose1,7-bisphosphate

2.5

mM

91

58

15 ml1

70

27

1 mM

38

26

remaining activity was compared same amount of protective agent

bEthanol equal no effect of

to that ethanol

cDEP was dissolved the preincubation

Although (data

there

not

in of

pH.

ethanol and furnished at 1.1 mM in all experiments.

faster

7.0 was chosen There

was also

(Tris

vation

(data

not

shown).

the

enzyme was preincubated

for

or phosphate)

1 or 10 minutes

did

To determine

substantial

at 30°C before

(Table

protection

tested.

Although

was substantially

DEP but

having

1) prior

there

solubility

of the

at pH 8.1. affect

with

The

the

inacti-

with

RuBP

RuBP, 3-phospho-D-

or carboxyhexitol-1,6-bisphosthe addition

inactivation

of DEP which

All

of these

by this

shown).

1094

compound

was present

compounds

by DEP at both

was some protection

higher

higher

of 6.2 and

of DEP interaction

at pH 7.0,

to assay.

against

during

at pH values

not significantly the site

was

concentration

inactivation

D-sedoheptulose-1,7-bisphosphate, 5 minutes

a final

of the

substantial

of buffer

glycerate,

lacking

inactivation because

nature

carboxylase,

with a control and ethanol.

furnished with DEP was added; however, there in comparison with controls lacking ethanol.

was somewhat

shown),

enzyme at this

tection

1 Min

mM

aThe the

times

Remaininga for

2.7

DEP + carboxyhexitol1,6-bisphosphate

phate

Activity Incubation

DEP + ribulose-1,5bisphosphate

DEP + 3-phosphoglrcerate

6.6

COMMUNICATIONS

the

showed

inactivation

by RuBP at pH 6.4, at pH 7.6

for

and 8.1

pro(data

not

Vol.

94, No.

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

0.0

0.15

I ;; E 0.1a ,” c !!!

1 I n 0”

0.6

= t :: I 0.4 -o

z

0 6

2 0.05

:

0.2

0.0

0.0

IO

20 Minutes

30

40

50

2.

increase

spectrum

in the

showed

increase

modification cm-')

reaction

per

the

histidine

absorption

maxima

absorptions

for

7.0,

residues

at 240 nm were

reversed

almost

by treatment incubation

residual

at 30°C in the

at 4°C of the

enzyme activity

coefficient

buffer 2).

to the

subunit.

However, of these

by the

The

the

incremental

addition

of NH20H

2). of 0.3 mM DEP could

NH20H.

enzyme with

of 55% was increased

1095

as at pH 7.0

at pH 6.4 also

Both

presence

enzyme with

DEP-modified

absorption

at 240 nm 3.2 x

and small

completely

of 0.4 M (Fig.

of the modified

This buffer

at 240 nm (Fig. was at 230 nm.

lost

shown).

of large

increase

concentration

The difference

at 240 nm corresponded

DEP in succinate

in absorption

2).

by a

in the ultraviolet

in phosphate

(extinction

combination with

Most of the activity

48-hour

enzyme,

this

at pH 7.0 and a final

recovered

untreated

same at pH 6.4

70,000-dalton

increase

at 240 nm (Fig.

in absorption

of M-acetylcysteine a marked

absorption

by DEP was accompanied

maxima at 240 nm (not

The increase of 2.4

RuBP carboxylase

enzyme versus

an absorption

buffer.

M-'

.3

OF INACTIVATION

differential

was essentially

in Tris

showed

of spinach

of DEP-treated

region,

lo3

00

Reaction of DEP with spinach RuBP carboxylase (1.2 uM) in 0.1 M and with II-acetylcysteine in 0.1 phosphate buffer, pH 6.4, -; M succinate buffer, pH 6.4, ------. The differential absorption at 240 nm was measured with a Cary model 14 spectrophotometer.

The modification sharp

70

Hours TIME

Figure

60

to 89%.

For example,

after

0.4 M NH20H at pH The same incuba-

be

Vol.

94, No.

tion

4, 1980

in the

BIOCHEMICAL

absence

of NH20H did

The enzymatic

activity

treatment

1 hour

for

DISCUSSION:

from

that

DEP is

indicate

that

DEP rapidly

The saturation

increase 2.4

with indicate

that

completely but

is

cysteine is

also

buffers.

not react

this

reversed

We do not

residues

for

by NH20H. think

that

maximum for

acetylcysteine (Fig.

is

present

buffer;

there

(2)

240 nm; and (3)

and DEP, in succinate

buffer

of about conclude

amino

DEP reacts

1096

under

is a sharp

and

(3)

of

the almost

though

with

at pH

N-acetyl-

in absorption

is

(6-7)

groups.

only

at 240 nm

in carboxylate is due to cysteine

no known reaction

is

of pH 6.4,

between

absorption

maximum

230 nm, whereas

of the

2 hrs compared

that,

pH range

there

groups,

derivative

the product

This

is also

the difference

the N-carbethoxyhistidyl

We therefore

activity

DEP modification

(1)

site.

to the modification

occurs

DEP and N-acetylcysteine

2) and has a half-life

imidazole.

the

suggest

site.

subunit;

an increase

The reaction

reasons:

between

that

protection

active

in the

corresponds one small

with

RuBP carboxylase

(2)

ethoxyformylated-NH2

causes

for

RuBP carboxylase)

with

reaction

in phosphate

absorption

histidine;

known to modify

have reported

the following

reaction

plus

(15)

DEP and cysteine the

for

(1)

by NH20H and enzymatic

DEP is also

and Himes

and that

specific

its

at the active facts:

RuBP

strongly

of spinach

of histidine

at 240 nm, which

reversed

NH20H does

at or near

inactivation

per one large

restored.

Garrison

which

highly

residues

increase

NH20H

sedoheptulose-1,7(12)

by the following

in the absorption

0.D240

4.0,

DEP is

along

inhibitors

RuBP carboxylase

is supported

histidine

similar

inactivates

kinetics

or the competitive

by DEP is due to the modification

of modification,

of the activity.

55% to 79% after

and carboxyhexitol-1,6-bisphosphate

further

interpretation

any restoration

COMMUNICATIONS

from

plant.

interacting

Our data

cause

RESEARCH

increased

results

a higher

(13)

BIOPHYSICAL

at 25°C.

by RuBP, 3-phosphoglycerate bisphosphate

not

was also

The present

carboxylase

AND

(and

the

DEP-modified

reaction

between

is much less to 55 hrs for

the conditions

h-

stable N-carbethoxy

described,

DEP

Vol.

94, No.

4, 1980

specifically for

catalysis

modifies

BIOCHEMICAL

histidine

by the carboxylase

AND

residues, activity

BIOPHYSICAL

one (or more)

RESEARCH

of which

COMMUNICATIONS

is

of RuBP carboxylase/oxygenase

essential from

spinach. ACKNOWLEDGMENTS: We thank Dr. Fazal R. Khan for the critical discussions during this work. This research was supported in part by grants from the Herman Frasch Foundation and NIH (GM-19,972). REFERENCES

:: 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Wildman, S. G. (1979) Arch. Biochem. Biophys. 196, 598-610. Hartman, F. C., Norton, I. L., Strinaer, C. D., and Schloss, J. V. (1978) in "Photosynthetic Carbon Assimilation" H. W. Sigelman and G. Hind, Ed., Plenum Press, New York, p. 245-269. Whitman, W. B. and Tabita, F. R. (1978) Biochemistry 17, 1282-1287. Purohit, K., McFadden, B. A., and Lawlis, V. 5. (1979rArch. Microbial. KJ, 75-82. Lawlis, V. 8. and McFadden, B. A. (1978) Biochem. Biophys. Res. Commun. @, 580-585. Schloss, J. V., Norton, L., Stringer, C. D., and Hartman, F. C. (1978) Biochemistry l7-, 5626-5631. Sugiyama, T., and Akazawa, T. (1967) J. Biochem. (Tokyo) 62, 474-482. Robison, P. 0. and Tabita, F. R. (1979) Biochem. Biophys. Res. Commun. 88, 85-91. Melchior, Jr., W. B. and Fahrney, D. (1970) Biochemistry 9, 251-258. Miles, E. W. (1977) in "Methods in Enzymology" Volume 47, Part E, C. H. W. Hirs and S. II. Timasheff, Ed., Academic Press, pp. 431-442. McFadden, B. A. (1973) Bacterial. Rev. 37, 289-319. Gordon, G. L. R., Lawlis, V. B., and McFadden, B. A. (1980) Arch. Biochem. Biophys. 199, 400-412. Saluja, Ashok K. and McFadden, B. A. (1978) FEBS Letters 9&, 361-363. Roche, T. E. and McFadden, B. A. (1969) Biochem. Biophys. Res. Commun. 37. 239-246. Garrison, C. K. and Himes, R. H. (1975) Biochem. Biophys. Res. Commun. 67, 1251-1255.

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