Synergistic inhibition of ATP phosphoribosyltransferase by guanosine tetraphosphate and histidine

Vol. 74, No. 1, 1977

BIOCHEMICAL

SYNERGISTIC

AND BIOPHYSICAL

INHIBITION OF ATP FHOSPHORIBOSYLTRANSFERASE BY GUANOSINE TETRAPHOSPHATE AND HISTIDINE David

P. I%rton

and Stanley

Department of Chemistry, Santa Barbara, California Received

RESEARCH COMMUNICATIONS

October

M. Parsons

University 93106

of California

24,1976

Summary. In the presence of partially inhibiting concentrations of the feedback inhibitor histidine guanosine-5'-diphosphate-3'-diphosphate becomes a potent inhibitor of the residual activity of ATP phosphoribosyltransferase from Salmonella typhimurium. Inhibition by guanosine tetraphosphate is higher order and slowly reversible. Inhibition does not occur in the absence of histidine. This effect constitutes an important control on the rate of histidine biosynthesis in response to the general aminoacid nutritional state of the cell. Introduction The first catalyzes

the

enzyme of L-histidine reaction

(E.C.2.4.2.17) catalytic

given

in eq.1. 1

from Salmonella conditions

(1)

biosynthesis

ATP phosphoribosyltransferase

typhimurium

containing

in microorganisms

is a hexameric

one allosteric

site

enzyme under for

histidine

on

+2

ATP f oPRibPP

each subunit centrations

the

of its

gene

probable

level

creases

Copyright All rights

manner

overlap

the inhibit

(2,3).

involvement

of growth

the alarmone

greatly

0 1977

site

the enzymatic

The enzyme is in regulation

limitation

(3).

High

con-

reaction

of great

in a

interest

of the histidine

be-

operon

by Academic in any

in concentration

Press,

Inc.

form reserved.

caused

by any limiting

(6) guanosine-'j'-diphosphate-3'-diphosphate , resulting

PRibPP, Abbreviations used are as follows: phate; PRibATP, N1-(5'-phospho-B-D-ribosyl)adenosine guanosine-5'-diphosphate-3'-diphosphate.

of reproduction

active

at

(4,5).

conditions

in bacteria,

PPi + BPRibATP

can totally

cooperative

Under

1

does not

of histidine

positively cause

(2) which

a Histidine

172

in decreased

aminoacid in-

synthesis

5-phospho-o-D-ribose-l-diphostriphosphate;

of stable

ppGpp,

BIOCHEMICAL

Vol. 74, No. 1, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Time. I minute per division Figure

Recently,

RIM (7). for

Dependence of assay curve shape on incubation and assay conditions. The ligands indicated parallel to each assay before the slash were present during incubation of the enzyme while those after the slash were present in the assay medium. Histidine (400 (LM) was present in all incubations and 350 PM ppGpp also was present in the incubations indicated. Histidine and ppGpp concentrations when present in the assay were 100 PM and 350 PM, respectively. Increased noise in assays containing ppGpp was due to a high absorbance background contributed by the guanosine chrcsnophore.

1.

expression

separable that

it

but

operon

histidyl-tRKA

ppGpp functions

Materials

shown that

of the histidine

from the

biosynthesis,

has been

also only

Kis

through

specific

as an inhibitor

in the

presence

ppGpp acts

as a positive

a mechanism mechanism

of the

first

which

(6).

effector is

We report

here

enzyme of histidine

of histidine.

and Methods

ATP phosphoribosyltransferase and the second and third enzymes of the histidine pathway were isolated and stored as previously described (8). ATP phosphoribosyltransferase was diluted 100 fold into pH 7.5 standard buffer (8) containing 0.1 mg bovine serum albumin/ml, 1 mM dithiothreitol, 0.40 mM histidine and 0.35 or 0.50 mM ppGpp when present and incubated 1 hr at 0' before assay. Assays were conducted as previously described (8) at pH 8.50 with the free Mg'2 concentration held at 2.0 mM utilizing a Cary 118 ultraviolet spectrophotometer at 25'. All assays contained excess inorganic pyrophosphatase (Boehringer/Nannheim) and second and third histidine pathway enzymes. Results Figure

1 shows typical

histidine

and ppGpp present

enzyme.

In all

fold

upon addition

cases

assay during

an identical

to the assay

curve

shapes

incubation

173

various

of the

concentration medium.

for

Thus

combinations

enzyme and assay

of of the

of enzyme was diluted ligands

present

during

120 in-

BIOCHEMICAL

Vol. 74, No. 1, 1977

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

I

,

-0 His -

0

Figure

2.

cubation the

of the

ligand

enzyme were

was not

already

2 contained

represents

the

fully

of ppGpp in the assay contained

no ligands

incubated

in the

enzyme activity minutes.

diluted

active but

showed

other

than

presence

lag

final

level

after

several

presence

in expression of activity minutes.

other

than

no effect substrates

of ppGpp plus

to zero

assay

Assay

enzyme.

sufficient Assay

substrates

of the

as in the absence Thus,

ppGpp plus

174

a high

on the enzyme in the

assay

histidine.

if

histidine

but

fully

in expression after

assay

to d except

but

2

that

again

to enzyme

A very the

of

several

solution

was assayed.

is apparent,

pro-

same

ppGpp was attained

of incubation histidine

Assay

the enzyme was

active

in the

and

concentration

activity.

A lag

4: was identical

activity

the assay

in the assay

2 contained

histidine

of ppGpp plus

during

solution.

The enzyme became

inhibition.

in the

in the

no ligands

Assay fi contained

incubated

effectively

present

is apparent.

35 percent

nounced

0.6

Guanosine tetraphosphate inhibition of ATP phosophoribosyltransferase in the presence of histidine. Enzyme incubated in the presence of 400 IJ.M histidine and 500 p,M ppGpp was assayed in the presence of the indicated concentrations of histidine and ppGpp. Maximal reaction velocity attained after recovery from any lag period is reported in all cases.

Assay

give

0.2 0.4 PPGPP. mM

in the

absence

of sub-

Vol. 74, No. 1, 1977

BIOCHEMICAL

TABLE: 1 - Guanosine

AND BIOPHYSICAL

Tetraphosphate

Apparent

Histidine Concentration (PM)

25

321

2.2

50

139

1.9

minutes

to activate

velocity

an inhibited

in the inhibition

f was greatly

in f suggested

time,

was established

essentially inhibitor

histidine totally

decreased. full

this

results.

solution

concentration

was not

upon exposure

in the

as shown

in the

since the

in assay

zero

time

downward

established

at zero

to ppGpp in the

of these

g.

in the

the

assay.

slight

enzyme incubated presence

and should

in the

assay presence

two ligands

was

Thus ppGpp is a total presence

of a moderate

of histidine.

The incubation synergistic

several

inhibitory

However,

inference,

and assayed inhibited

assay

established

inhibition

progressively with

requires

ppGpp to become an inhibitor

of ATP phosphoribosyltransferase

concentration

the

that

In agreement

of ppGpp plus

induced

in the

of a partially

by ppGpp was rapidly

curvature

solution.

histidine

The presence

assay

enzyme which

of replots of Fig.1 in the is the velocity in its presence.

of ppGpp.

ppGpp plus

to b and -d.

from the slopes of histidine in ppGpp, where vi is the velocity

form of the

upon removal

2 contained

of assay

but

1.6

76

produces

Substantial

Apparent PPGPP Hill Coefficient"

co

strates

of histidine

Constants

0

aHi. coefficients were obtained the data for each concentration form log Vi/(V-Vi) versus -log in the absence of ppGpp and Vi

be compared

Inhibition

Apparent PPGPP Constant (PM)

100

Assay

RESEARCH COMMUNICATIONS

conditions

relationship At increasing

of assay g then

between

were

ppGpp and histidine.

concentrations

175

of histidine

adopted Figure lower

to explore

the

2 presents concentrations

Vol. 74, No. 1, 1977

of ppGpp are enzymatic (Table

BIOCHEMICAL

required

activity. I).

(Fig.2,

1 lists

residual

78 PM ppGpp is required of histidine

25 PM, ppGpp can still

tetraphosphate Hill

of about

histidine

only

of the

insufficient

to

be an effective

inhibitor

1).

Guanosine

values

of 50 percent

at a concentration

by itself,

Table

inhibition

At 100 PM histidine

Furthermore,

show inhibition

Table

to obtain

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inhibition

coefficients

obtained

1.6 to 2.2

Hill

is a positively

are

coefficient

from

similar

cooperative

separate

to those

Hill

often

process.

plots.

obtained

The

for

the

(2).

Discussion The observation transferase that

that

in the

binds

to this

presence

of histidine

versatile

enzyme.

histidine

the

histidine

operon

it

be determined

cannot

binding

site

enzyme also

the

constant

inhibition

ATP phosphoribosyla new class

In addition

to

the

would

Even though

data

presented

binding

of substrate

here

His

whether If

it

(4),

higher

the

and

inhibitor ppGpp

does the apparent

of substrate

causing

and

ppGpp is a total

sites.

be a function

of ligand

substrates

histidyl-tRNA

AMP and ADP (3),

substrate

concentrations

inhibits establishes

DNA (4). from

overlaps

higher

concentration,

apparent

ppGpp

constants. Although

the

ppGpp inhibition these

binds

attenuator

ppGpp inhibition with

ppGpp reversibly

data

simplest

is

do not

that

bound

prove

this

the

enzyme in the

absence

the

ppGpp binding

site

histidine

could

ppGpp inhibitory cooperative

of the

histidine model.

creates Guanosine

of histidine

was distinct

but from

a conformational

and promoted

further

not

histidine the

reversible

ppGpp binding

tetraphosphate affect

the active change

requirement site,

could

enzymatic site.

bind

activity

Binding

in the enzyme which binding

for

to if

of made bound

of ppGpp in a

manner.

Inhibition and substrates

cause

interpretation

by ppGpp is effective which

are

comparable

at concentrations to estimated

176

--in vivo

of histidine, levels

for

ppGpp, bacteria

BIOCHEMICAL

Vol. 74, No. 1, 1977

growing the

in glucose-minimal

following acid

was not

since

increases

consequences

for

histidine,

histidine

histidine

The increase

histidine

and ppGpp inhibit manner,

inhibited.

Thus the general

metabolite

level

over

the

would

stringent

by means of ppGpp and histidine

rise bio-

with Since

an both

a positively

be very

state

of histidine

protein

coincide

in would

aminowould

response.

nutritional

rate

limiting

due to slowed

ATP phosphoribosyltransferase

control

have the

concentration

concentration

aminoacid

any aminoacid

should

ATP phosphoribosyltransferase

cooperative

important

as rapidly

of ppGpp due to the

for

If the

histidine

in histidine

concentration

This

(7).

biosynthesis.

not be utilized

increased

exert

greatly

the intracellular

could

synthesis.

Upon starvation

(6,9).

salts

ppGpp concentration

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the

biosynthesis

effectively cell

could

at the

synergism.

Acknowledgement This

work

was supported

by U.S.

Public

Health

Service

grants

GM 20835 and

GM 23031. References 1.

Parsons,

S. M.,

and Koshland,

D. E.,

Jr.

(1974), J. Biol.

4119-41~26. 2. 3. 4.

Chem. 249 -2

Bell, R. M., Parsons, S. M., Dubravac, S. A., Redfield, A. G., and Koshland, D. E., Jr. (19741, J. Biol. Chem. 249, 4110-4118. Martin, R. G. (1963), J. Biol. Chem. 238, 25-68. Goldberger, R. F., and Kovach, J. S. m72), Curr. Top. Cell. Reg. 2,

285-308; 6.

Scott, J. F., Roth, J. R., and Artz, S. W. (1975), Proc. Natl. Acad. Sci. (US) 72, 5021-5025. Stephens, J. C., Artz, S. W., and Ames, B. N. (1975), Proc. Natl. Acad.

7.

;;hel(US; 72, 4389-4393. J. , ., and Gallant,

5.

8.

(1974), in Ribosomes, eds., M. Ncmura, A. Tissieres, and P. Lengyel, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., PD .733-745. Morton; D. P:,--&d-Parsons, S. M. (19761, Arch. Biochem. Biophys. 175 2 677-686.

9.

Brenner, M., and Ames, B. N. (19711, in Metabolic D. M. Greenberg and H. J. Vogel, Academic Press,

177

Pathways New York,

V, eds.,

pp.349-387.