Tryptophan-mediated substrate inhibition of anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase

Vol. 35, No. 1, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TRYPTOPXAN-MEDIATED SUBSTRATE INHIBlTION OF ANTHRANIL4TE-5PHOSPHORIBOSYLPYROPHCWHATE PHGSPHORlBOSYLTRANSFSEl H. Zalkin and Ellen J. Henderson2 Department of Biochemistry, Lafayette,

Purdue University,

Indiana 47907

Received February 27, 1969

A previously unrecognized type of endproduct inhibition is described. Anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase (PR transferase) activity from Salmonella typhimurium when aggregated to active or inactive anthranilate synthetase, is subject to endproductmediated substrate inhibition. Inhibition of PR transferase activity by the substrates, 5-phosphoribosyl-1-pyrophosphate (PRPP) and anthranilate, is observed in the presence of tryptophan. Substrates for the aggregated anthranilate synthetase enzyme antagonize the tryptophanmediated substrate inhibition of PR transferase activity. Tryptophan synthesis in Salmonella typhimurium (l), coli

(2,3) and Aerobacter aerogenes (4) is regulated,

endproduct inhibition

of anthranilate

in part,

These two enzymes which catalyze

of tryptophan anthranilate

synthetase and PR transferase

inhibition

(PA transferase)

the first

synthesis are aggregated in wild-type

from appropriate mutant strains.

two reactions

cells.

activities

Whereas PR transferase

from anthranilate

to endproduct inhibition

synthetase.

Unaggregated

can be obtained

by tryptophan when aggregated to anthranilate

the enzyme is insensitive

by

synthetase and anthranilate-y-

phosphoribosylpyrophosphate phosphoribosyltransferase activities.

Escherichia

is subject to synthetase,

when dissociated

The reactions catalyzed

by anthranilate

1 Journal Paper No.3(33 Purdue Agricultural Experiment Station. Supported in part by Grant GB-5841 from the National Science Foundation. 2 National Science Foundation predoctoral fellow.

52

Vol. 35, No. 1, 1969

BIOCHEMICAL

synthetase and PR transferase Pyruvate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are shown below.

Chorismate Mg2+ Anthranilate Mg2' >PR anthranilate+ A Glutamine Glutamate PRPP PPi

+ +Tryptophan

The purpose of this paper is to describe experiments which elucidate, in part,

the mechanismof endproduct inhibition

tryptophan.

of PR transferase by

It is found that tryptophan mediates substrate inhibition

of PR transferase

activity. METHODS

Two types of PR transferase were studied.

PR transferase,

obtain-

ed from S. typhimurium strain -trp E2, is aggregated to catalytically active

anthranilate

synthetase.

The enzyme obtained from S. typhimurium

strain trp A8 is aggregated to inactive -

anthranilate

synthetase.

Both

strains were grown under conditions that allowed derepression of the tryptophan

operon.

The native enzyme aggregate from strain trp E2 -

was purified

&fold

anthranilate

utilized/min./mg

(0 - 3% saturation), G-200 gel filtration. purified

19-f&3

utilized/min./mg saturation)

to a specific

activity

of 2540 rnp moles

protein by (NH42) SO4 Precipitation

DEARcellulose

chromatography and Sephadex

The enzyme aggregate from strain -trp A8 was

to a specific

activity

of 635 m)l moles anthranilate

protein by ammoniumsulfate

precipitation

and DRABcellulose chromatography.

(0 - 40$

Both enzyme aggregates

have a molecular weight of approximately

23O,OCO(g20 w = 10.7s) as , determined by sucrose gradient centrifugation (5), using yeast alcohol dehydrogenase (6.7s)

as a marker.

PR transferase

activity

mined at room temperature (22') by spectrofluorometric rate of anthranilate triethanolamine-HCl

utilization.

was deter-

assay of the

Reaction mixtures contained 0.05 M

pH 7.5, 20 nJ45-phosphoribosyl-1-pyrophosphate

(PRPP), 20 nI4 anthranilate,

2 mMMgC12, 1 mMdithiothreitol

53

and a

Vol. 35, No. 1, 1969

limiting

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

amount of enzyme. Deviations

from this procedure are speci-

fied where applicable. RESULTSANDDISCUSSION As reported previously

(2), PR transferase

typhimurium is inhibited

by tryptophan.

shows that at saturating

concentrations

(20 @ each) inhibition inhibition

by tryptophan

activity

from S -*

The upper curve in Figure 1 of PRPPand anthranilate approaches 7%.

Half maximal

is obtained at less than 1.0 @! tryptophan.

In order

0 0

/

TRYPTOPHAN

pM

of PR Figure 1. Effect of tryptophan concentration on inhibition transferase activity at saturating and limiting concentration of substrate. The reaction mixture is as described in Methods except as noted. The symbols arem---- m, 20 p.Manthranilate and PRPP; 0 -0, 2 @l anthranilate and 20 @I PRPP; l -- l , 20 pM anthranilate and 1 pM PHPP Enzyme is from strain trp A& Negative values for inhibition denote activation, to determine the type of inhibition of varying

the concentration

exerted by tryptophan,

of one substrate at fixed concentrations

of the second substrate was studied.

In Figure 2A a plot of l/v

against l/PRPP is shown. In the absence of tryptophan linear

double reciprocal

tryptophan

plot is obtained.

cause substrate inhibition,

at high PRPPconcentrations

the effect

the usual

However, 2 ELM or 20 pM

as shownby upward curvature

and apparent convergence at low PRPP

54

BIOCHEMICAL

Vol. 35, No. 1, 1969

1.5

r

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A 60

00,

0.5 I / PRPP

1.0 ,u hi’

1.5

‘0

4

8 PRPP

12 16 20 ,LLLM

Figure 2. Effect of PRPPconcentration on tryptophan-mediated inhibition. (A) Double reciprocal plot. (B) Samedata plotted as The symbols are e-----e, no $ inhibition against PRPPconcentration. tryptophan; o--o, 2 ClMtryptophan;&---¤ , 20 @l tryptophan. Enzyme is from strain -trp A8.

The dependence on PRPPfor tryptophan-mediated

concentrations. inhibition

is shownmore clearly

Figure 2A have been replotted PRPPconcentration. phan is clearly

in Figure 2B where data from

in the form of % inhibition

Inhibition

a function

of PR transferase

of the PRPPconcentration.

Figures 3 and 3B show the samequalitative saturation

by anthranilate.

tryptophan, observed.

inhibition

relationships

for

In the presence of 2 @l or 20 @l

of PR transferase

activity

by anthranilate

is

of anthranilate

and was not observed in this experiment.

Since tryptophan inhibition

causes substrate inhibition

concentration

of anthranilate

PRPPconcentration rather

of PR transferase

should'be reduced at low substrate concentrations,

The data in Figure 1 show reduced inhibition

inhibits,

by trypto-

The data in

Convergence of the curves at low concentrations

is variable

activity,

activity

against

by tryptophan when the

is lowered from 20 ~ to 2 @& When the

is lowered from 20 m to 1 @l, tryptophan no longer slight

activation

phan at low PRPPconcentrations

is observed.

is variable.

55

Activation

by trypto-

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 35, No. 1, 1969

B

60-

000.5

.

1.5 '0

I /ANTHRANILATE

,uM-’

4

8

12 16 20

ANTHRANILATE

,uM

Figure 3. Effect of anthranilate concentration on tryptophan-mediated inhibition. (A) Double reciprocal plot. (B) Samedata plotted as The symbols are given in Figure 5 inhibition against PRPPconcentration. 2. Enzyme is from strain trp A& -

80

'0

20

40

CHORISMATE

60

80

B

t

200

too

400

CHORISMATE

,uM

600

800

pM

Figure 4. (A) Effect of chorismate on tryptophan-mediated substrate inhibition with wild-type enzyme. (B) Effect of chorismate plus glutsmine on tryptophan-mediated substrate inhibition with enzyme from strain trp A& The glutamine concentration is 5.0 mM. The abbreviation?% tryptophan is trp.

Although the data in Figures 1-3 only show results ferase from strain tively

similar

trp A8 (inactive -

anthranil.ate

synthetase) qualita-

data are obtained for PR transferase

56

for PR trans-

from strain -trp

BIOCHEMICAL

Vol. 35, No. 1, 1969

E2 (active

anthranilate

synthetase).

Tryptophan-mediated is

antagonized

synthetase

effect

substrate

by the

on the

exerts

plus

enhances a stronger

for

Figure

native

of chorismate

Glutamine

inhibition

substrates

Data in

enzyme.

of chorismate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

glutamine

aggregate

that

compared

to the

aggregate

We suggest

effect

of chorismate

of PR transferase is

synthetase

(3).

(b)

change

Substrates

which

substrate

activity

renders

substrate

synthetase is

with

an inactive to account

The only

tryptophan

competitive

for

substrate

Inhibition

synthetase

~8.

Chorismate an active

enzyme.

anthranilate

of anthran-

with

induce

binding

chorismate

a conformational

to endprcduct-mediat&

insensitive

physiological

is

that

of prephenate

the NAD concentration concentrations

Prephenate

in the

activity

(6).

suggest

dehydrogenase in

tyrosine

dehydrogenase Inhibition

of NAD and is

data

enzyme activity

of endproduct-media-ted is

presently

uncertain.

are in progress.

of interest

These

significance

of PR transferase

experiments

inhibition

NAD.

(a)

Pli transferase

inhibition

Further

high

trp -

inhibition.

The potential

It

strain

on tryptophan-mediated

by tryptophan for

antagonistic

contains

two explanations

activity.

on the anthranilate

ilate

of chorismate.

on the

synthetase.

site(s)

effect

effect

anthranilate

inhibition

enzyme from

antagonistic

effect

4B the

on the

the

activity

anthranilate

antagonistic

enzyme and in Figure

synthetsse

antagonistic

aggregated

LA show the

anthranilate

the

the

of PR transferase

pathway

by tyrosine

is

dependent

upon

by tyrosine

is

strongest

at

minimal

at low concentrations

endproduct-mediated activity

the pathway,

is

in A. aerogenes,

coenzyme

associated

chorismate

with

mutase

of

inhibition. the preceeding

(6,7).

ACKNOWLEDGEMENT Expert Discussions

technical with

assistance Stanley

W. Artz

was provided were

57

by Mrs.

instrumental

Lian-Hua to this

Hwang. work.

Vol. 35, No. 1, 1969

We are grateful

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

to Dr. R. Bauerle for supplying 5. typhimurium strain

trp A8. REFERENCES 1.

2. 3.

4.

2: 7.

Bauerle, R. H., and Margolin, P., Cold Spring Harbor Symp. Quant. Biol. 3l, 203 (1966). Moyed, 'I. S., and Friedman, M., Science 12q, 968 (1959). Baker, T. I., and Crawford, I. P., J. Biol. Chem.2&, 5577 (1966). Egan, A. F., and Gibson, F., Biochim. Biophys. Acta 130, 276 (1966). Martin R G and Ames B. N J. Biol. Chem. 2$, 1372 (1961). Cotton' R' G:'H., and Gfbson,'; ., Biochim. Biophys. Acta 147, 222 (1927): Cotton, R. G. H., and Gibson, F., Biochim. Biophys. Acta 100, 76 (1965).

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