Inactivation of bean ornithine carbamoyltransferase by phaseotoxin: Effect of phosphate

Vol. 89, No. 4, 1979

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

August 28, 1979

Pages 1361-1368

INACTIVATION OF BEAN ORNITHINE CARBAMOYLTRANSFERASE BY PHASEOTOXIN: EFFECT OF PHOSPHATE Oliver

C. H. Kwok',

Harry

Ako* and Suresh

S. Patill

Departments of Plant Pathology' and of Agricultural Biochemistry*, University of Hawaii, Honolulu, Hawaii 96822 Received

July

16,1979 SUMMARY

The results of kinetic studies of the inactivation of bean ornithine carbamoyltransferase by phaseotoxin, the extracellular toxin of Pseudomonas phaseolico&, are consistant with the notion that the toxin is an active site directed irreversible inhibitor of the enzyme. Phosphate, an end product of the enzymatic reaction, protects the enzyme from inactivation by the toxin. It is proposed that phaseotoxin is one of a few naturally occurring affinity labels.

INTRODUCTION Previous Pseudomonas beans,

is

beans.

studies

(1,2)

phaseolicola

a specific

Recently,

carbamylation

have shown that

(Burk.)

Dowson,

and potent we found

of ornithine,

the causal

inhibitor

that

phaseotoxin agent

of ornithine

orthophosphate,

protects

(3)

the

the

exotoxin

of the halo

of

blight

carbamoyltransferase

which

enzyme from

is

released

of of

during

the

inactivation

by phaseotoxin

of inactivation

of ornithine

at low concentrations.

In this

communication

carbamoyltransferase phosphate.

that

phosphatefphosphate

enzyme from the

the site

are consistent

number of naturally

for

by the

and its

protection

the

equations

derived

toxin

is an affinity

of the with

affinity

by phosphate

enzyme can be explained

label

enzyme and which

the notion

occurring

inactivation

the kinetics

toxin

We have employed

the hypothesis

results

we examine

that labels

inactivation

by Meloche which

and that high

consequence

(4)

binds

irreversibly

phaseotoxin

and the very

as a natural

from

by to examine

to the carbamoyl inhibits

it.

The

is one of a small the protection potency of this

of the

of phaseotoxin fact.

0006-291 X/79/1 61361-08$01 .00/O 1361

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 89, No. 4, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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MATERIALS AND METHODS Preparation of Enzyme and Toxin prepared as previously desczem.

- Ornithine

carbamoyltransferase

was

Phaseotoxin was isolated from innoculated bean primary leaves as described previously (6) except that ultrafiltrates were chromatographed on a 1.5 x 102 cm column of Sephadex LH-20 developed with deionized water. Phaseotoxin is a family of closely related toxins (6) which may or may not contain a toxin which has been purified from another isolate of p. phaseolicola (7) using different purification and bioassay procedures. The partially purified phaseotoxin was used in the present work because of the unavailability of any one component of phaseotoxin free of degradation products. One unit of toxin is defined as that amount of toxin which reduces the activity of 0.05 unit of enzyme by 50% under assay conditions described below. Determination of Rate of Inactivation. Kinetic studies of toxin inactivation of ornithine carb=ltransferase were conducted at 30°C and pH 8.5. The reaction mixture contained 25 pmoles of iminodiethanol, 5 pmoles of-L-ornithine, various amounts of toxin, and water to a volume of 0.8 ml. The inactivation reaction was started by adding 0.05 unit of enzyme in a volume of 0.1 ml to the reaction mixture. At various time intervals, 5 pmoles of carbamoyl phosphate in a volume of 0.1 ml was added to stop the inactivation reaction. This would be sufficient to prevent further inactivation since toxin causes no inactivation in the presence of 5 pmoles of carbamoyl phosphate. The addition of carbamoyl phosphate also started the enzymatic reaction. Incubation was allowed to proceed for another 10 min and the reaction was stopped by adding 5.25 ml of Citrulline formation was determined spectrophotometrichromophoric reagent. tally as previously described (5). Irreversibility of Inactivation. Aliquots of enzyme were incubated at 37'C for 20 min with z without toxin (100 units of toxin per unit of enzyme). The samples were dialyzed against 1OmM Tris-HCl buffer, pH8.5, overnight at 4'C before enzyme activity was determined. RESULTS Inactivation carbamoyltransferase shown in Fig was increased. toxin

at three The rate

1.

clearly

of inactivation extrapolations half-times

in Fig

of inactivation

concentrations

(1.5,

increased

2 which

incubation is

as the

rates

time

is

Fig

2 also

toxin

allowed. plot

at higher This

is

of the time

demonstrates

of inactivation

is

concentration

completion

a semilogarithmic

of toxin.

of ornithine 3.0 and 6.0 units)

of the enzyme approaches

when sufficient

initial

course

of inactivation

by 3.0 units of the

that

course

linear

can be made to determine

of inactivation.

According non-covalent

toxin

The inactivation

concentrations

demonstrated

The time

Kinetics.

to the model enzyme-inhibitor

developed complex

by Meloche would

1362

(4)

be formed

for

affinity

prior

labels,

to labelling

a of the

Vol. 89, No. 4, 1979

BIOCHEMICAL

AND

I

0

4

1

8 Time

BIOPHYSICAL

02

12

(Mid

of inactivation Fig. 1. Time-course phaseotoxin at three different toxin (0) per ml. (4 > and 6.0 units

RESEARCH

4

12

8 Time

of ornithine concentrations:

COMMUNICATIONS

Knin)

carbamoyltransferase 1.5 units (O),

by 3.0 units

Fig. 2. Time-course of inactivation phaseotoxin (3 units/ml) presented shows the rapid phase (pseudo first

of ornithine carbamoyltransferase by in a semi-logarithmic plot. The dashed order phase).

enzyme by the

would

toxin.

Inactivation

proceed

E+I&EI+EI where E represents complex

and EII

following

free the

{equation

enzyme,

to equation

EI the Michaelis-type

inactivated

1

(1)

I

, toxin,

irreversibly

according

line

enzyme.

enzyme-toxin

From equation

1, the

can be derived

(2) where

t, r'epresents -5

saturating

toxin

inactivation concentration

half-time, and K. 1'

the

T$, toxin

the

inactivation

binding

constant.

half-time

at

Thus a plot

of toxin concentration should give a straight Q,2 versus the reciprocal line with ,a slope of TQKi and an intercept at T, . When half-times of inactiva-5 tion are plotted as a function of reciprocals of toxin concentrations the data

of

1363

Vol. 89, No. 4, 1979

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5-

4-

-2 .-E zN

3-

2-

l-

/ I

c3

0.4

02 l/Toxin

3

4

0.6

12

6

(U-‘)

Time

(m!n)

Fig. 3. Inactivation half-time as a function of the reciprocal of phaseotoxin concentration. The values of t were determined from extrapolations similar to 4 those shown in Fig 2. Fig. 4. Effect of phosphate ions on inactivation. with 6 units of toxin and various concentrations of 0.0 m M (0), 0.2 m M (A) and 0.6 m M (0). The ordinate activity remaining expressed as a percentage of the priate controls were used to correct the inhibition (5% inhibition at 0.6 m M phosphate). in Fig

3 are obtained.

line.

A value

The experimental

of 0.1 min for

Irreversibility

points

T4 and 63.1

-of Inactivation.

showed only

an activity

dialysable,

seems to form a stable

complex

If

phosphate

Protection with

phase of toxin

equat .on 1 can be expanded

binding

describe

a straight

Ki were obtained. the

dialysis, Therefore,

of 3% of the control.

by Phosphate.

the reversible

clearly

U for

After

Enzyme was pre-incubated phosphate before assay: represents the enzyme initial activity. Approcaused by phosphate alone

with

toxin the

treated

toxin,

binding and if

to the toxin

enzyme is

were an affinity

I

+ (3)

1364

is

the enzyme.

to E+IeEI---rEI

which

enzyme

competitive label,

Vol. 89, No. 4, 1979

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

>-

/ Z

I

0.2

0

6

0.4 Phosphate (mM)

0.6

Fig. 5. Effect of phosphate ions on inactivation as shown in a plot versus reciprocal of toxin concentration. Phosphate concentrations: (O), 0.1 m M (A) and 0.5 m M (0). of phosphate Fig. 6. Effect phosphate concentration plot. concentrations of phaseotoxin where Pi represents shows the units

time

of toxin

course

rate

where I represents K

P' study

the phosphate in which

of ornithine

and the presence decreased

complex.

Fig

carbamoyltransferase

4 by 6

of 0.2 and 0.6 m M phosphate.

when the enzyme was treated

with

of phosphate.

relationship

tl

and EPi the enzyme-phosphate

was greatly

in the presence The following

ions on inactivation as shown in a tr, versus Explanations are given in the text. Two were used: 4 units (0), and 6 units (A) per ml.

of inactivation

in the absence

The inactivation toxin

phosphate

of tl 0.0 i&l

5

can be derived

= -&C,i.+--)+T

toxin, binding

t 4 is plotted

toxin

constant.

3:

K. [PiI P

Ki,

from equation

binding Fig

as a function

1365

4 constant,

P i,

5 shows the results of the reciprocal

phosphate

ion,

and

of the kinetic of toxin

concen-

Vol. 89, No. 4, 1979

tration

in

the absence

inactivation

and in

half-time

the plots with

BIOCHEMICAL

obtained

increasing

consistent

phosphate

test,

A plot

of t,

the slope Fig

versus

5

and the

intercept

protection

P concentration

of half-time

of ornithine

all

slope

plots

of increases

extrapolate

to

should

be influenced

versus that

as follows:

T K. + (TG + + ) 2

) [Pi]

should

per ml and demonstrates

As expected

The

The linearity

3 and 4, and the

4 can be rearranged

( >

phosphate

6 shows a plot

toxin

2

COMMUNICATIONS

saturating.

equation

t, = -i

by phosphate.

equations

concentrations. is

RESEARCH

of 0.1 mM and 0.5 mM phosphate.

increased with

when toxin

As a further

BIOPHYSICAL

the presence

was greatly

is

the same half-time

AND

a straight

by the

phosphate

equation

give

toxin

line.

Both

concentration.

concentration

at 4 and 6 units

5 can be used to describe

the

carbamoyltransferase.

DISCUSSION A previous reversible. tion. did

report However,

For instance, not

(5)

had suggested

other

observations

under

obey classical

certain

double

experimental

reciprocal

kinetics. toxin.

on preincubation

of enzyme with

show that

the

of ornithine

follows

kinetics

irreversible phosphate,

that

the

bind

toxin

binds

which

indicates

has no effect

to the active that

of the enzyme from

of the enzymatic

reaction,

carbamoyltransferase

to the

same part

with

In the presence

toxin

protection

ornithine

the

the

consistent

its

conditions Also,

inactivation

of the active

site

active

1366

This

here

site

of carba-

piece

directed

inhibitor would

phosphate.

directed

may indicate

Phosphate

phosphate

as carbamoyl

site

An additional

by phosphate.

Therefore,

presented

by phaseotoxin

and was shown to be a competitive (8).

of enzyme

concentrations

of the enzyme. is

sugges-

was

as an active

of saturating

inactivation

this

inhibition

The results

acting

may be

inhibition

on the enzyme (5).

site

complex with

carbamoyltransferase

are consistent

inhibitor.

moyl

evidence

which

the enzyme toxin

were not

dependent

inactivation

that

is is

of the

a product of

be expected

to

BIOCHEMICAL

Vol. 89, No. 4, 1979

Inhibition suggesting

could

that

of ornithine a model

not

ornithine

carbamoyl

a loose

for

by phaseotoxin

between

the

two,

is

kinetically

against

consistent

that

the

of inactivation with

enzyme and toxin

indicating

complex

inactivation

In particular,

the kinetics

between

the

was tested

labels.

that

COMMUNICATIONS

enzyme toxin

More rigorously,

affinity

complex

RESEARCH

of the

by phaseotoxin

transferase

reaction

by dialysis

3 demonstrates

Michaelis-type

BIOPHYSICAL

irreversible.

byMeloche(4)

shown in Fig

covalent

is

carbamoyltransferase

linearity

that

be reversed

inhibition

developed

AND

the notion

is

phaseotoxin

of

formed is

prior

to

an affinity

label. In equation formation

3, it

is proposed

of the Michaelis-type

consequences

of equation

of the plots

shown supports

interaction

of toxin

3 and 5 are mutually are the same in

several

The covalent in

binding

toxin its

high

Ki.

formation becoming

other

that

appears

in

form Thus,

only

inducing

being

T.,

of affinity

rhizobitoxin

complex.

(9)

labels produced

Moreover,

puzzling

why the

Inspite with

great

of T4

complex

inactivating potency

the by

the

and subsequently of the Michaelis-type

chlorotic

symptoms after

S. S.).

Phaseotoxin

appears

produced

by phytopathogenic strains

effective

can prevent

immediately

by certain

by

the enzyme as judged

of phosphate

from

is

and in

phosphate

1367

Figs

observations.

toxin

of its

The looseness

the relief

and Patil,

on the

as judged

in bean leaves

complex

toxin-enzyme

with

The linearity

the values

label

previously

(1,5).

concentrations

explains

kinetic

3.

that

the

of phosphate.

chlorosis

non-covalent

are treated

Oguchi,

two examples

moderate

also

demonstrate

several

non-covalent

in

of phosphate

equation

to the enzyme explains

toxin-enzyme

complex

when plants

explains

toxin

5 and 6.

effect

to be an affinity

--in vitro

a strong

the with

they

and absence

This

amounts

a covalent

(unpublished, only

consistent

of toxin

of the weak,

toxin-enzyme plants

consistent

carbamoyltransferase

does not

that

with

Moreover,

as shown in Figs

and enzyme is in

competes

complex.

the conclusion

criteria.

sub-microgram

ornithine

3 were tested

phaseotoxin

kiinetic

phosphate

toxin-enzyme

the presence

In summary,

that

of bean

innoculation to be one of bacteria,

of Rhizobium

the

japonicum.

Vol. 89, No. 4, 1979

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS We thank Professor Richard Guillory, Department of Biochemistry and Biophysics, John Burns School of Medicine, University of Hawaii, for a critical review of the manuscript. This work was supported by NIH Grant A109477. Hawaii Agricultural Experiment Station Journal Series Number 2422. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Patil, S. S., Tam, L. Q., and Sakai, W. S. (1972) Plant Physiol. 49, 803-807. (1970) Plant Physiol. Patil, S. S., Kolattukudy, P. E., and Diamond, A. E. 46, 752-753. 12, 259-279. Patil, S. S. (1974) Ann. Rev. Phytophathol. (1967) Biochemistry 6, 2273-2280. Meloche, H. P. Tam, L. Q., and Patil, S. S. (1972) Plant Physiol. 49, 808-812. Patil, S. S., Youngblood, P., Christianson, P., and Moore, R. E. (1976) Biochem. Biophys. Res. Commun. 69, 1019-1027. Mitchell, R. E. (1976) Nature 260, 75-76. Ravel, T. M., Grona, M. L., Humphreys, J. S., and Shive, W. (1959) J. Biol. Chem. 234, 1452-1455). Giovanelli,J.,Owens, L. D., and Mudd, S. H. (1971) Biochem. Biophys. Acta 227, 671-684.

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