Induction of the allantoin degradative enzymes by allophanic acid, the last intermediate of the pathway

BIOCHEMICAL

Vol. 52, No. 1,1973

INDUCTION OF THE ALLANTOIN

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DEGRADATIVE ENZYMES BY ALLOPHANIC ACID,

THE LAST INTERMEDIATE

OF THE PATHWAY

Terrance G. Cooper and Robert Lawther Department of Biochemistry, F.A.S. University of Pittsburgh Pittsburgh, Pennsylvania 15261

Received

March

16, 1973

SUMMARY Sacchahomyced c~&Xaiae can utilize allantoin as a sole nitrogen source by degrading it in five steps to ammonia, "CO2", and glyoxylate. We have previously shown that allophanic acid is the inducer of the urea carboxylase: allophanate hydrolase multienzyme complex. Since these enzymes catalyse the last two steps of allantoin degradation, experiments were performed to determine if allophanate was also the inducer of any other enzymes in the pathway. Our data demonstrate that allophanate induces synthesis of at least five of the seven purine degradative enzymes. Saccmmyced allantoin, tion

ce&u&&

allantoic

for

this

degradative

acid,

activity

function

was performed

ing

carboxylase

of others

these

urea,

arginine

found

(8)

constitutive in the basal

level

presence level

of urea; type

strains,

urea

analogue,

minus

strain,

(3-7)

source

Levenberg's

provided

to avidin

evidence

insensitive

(1).

demonstration

complex,

of urea

of urea,

formamide but would

only

that

composed

when using An explanathat (2).

this

a urea Our ex-

degradative

of a biotin

allophanate

in cultures

we sought

strains

lacking

the

hydrolase

whereas

and hydantoic not

serve

hydantoate,

Copyright 0 I973 by Academic Press, Inc. AN rights of reproduction in any form reserved.

identity

whether

strains

hydrolase

grown

hydrolase

contain(reac-

acid

did

of inducing

an allophanate 137

inducer.

We

possessed

a high

was grown

urea

carboxylase

produced

not

increase these

sources; the

of

the culture

induced

as nitrogen

presence

or not

lacking

which

in the

of their

allophanate

carboxylase

was not capable whereas

nitrogen

was sensitive

are found

of allophanate (2)

of biotin

1).

or a purine, (1)

in

m

and an avidin

enzymes

that:

quantities

as sole

by a multienzyme

4 and 5 in Fig. Since

large

was found

in Candida

and those

tions

or urea

observation

periments

urea

requires

upon the

activities and (3)

hydrolase analogue,

a

addition in wild

formamide,

a

in a carboxylase was able

to induce

BIOCHEMICAL

Vol.52,No.1,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PURINES----ALLANTOlN ‘1 ALLANTOATE

PROTEIN

UREIDOGLYOXYLATE

ORNliHlNE

)

ALLOPtiANATE

t 2NH3

Figure

1.

Reactions involved in the degradation in Sacchahomyces cehev~&z.

the hydrolase urea

in such a strain.

carboxylase

allophanic Although

also

a product

urea

(Fig. the

of the

allantoin.

The inducer

be arginine

(10).

The data

last

intermediate

phanate,

the

at least

five

last

In view

inducer

carboxylase

of these

other

of arginase

support

enzymes

unique

of

of as they

of arginine

was pertinent

to

in the degradation

6 of Fig.

here

of

presence

nitrogen,

it

participating

of the degradative

of the seven

of urea

in the degradation

(reaction

presented

production

may be thought

considerations,

enzymes

the

upon the

hydrolase

two steps

and allantoin

reaction.

in the assimilation

as the

1).

that

is contingent

and allophanate

functioning

may be considered

identify

of the urea

of arginine

data suggested

hydrolase

carboxylase

activities

and purines

These

and allophanate

acid,

anabolic

+ 2 “C02”

the

1) has been shown to contention

pathway

to purine

of

that

is the

allo-

inducer

of

degradation.

METHODS Data substantiating this

work

diploids, ments except

appears

biochemical

elsewhere

homozygous were

the

conducted

the concentration

for

(4,7,9). the

using

All

significant

a protocol of

phenotypes

(NH4)2S04

of the strains alleles.

similar

were

strains

to that

reported

was 0.1%.

used in

prototrophic

The physiological

in the media

138

of mutant

experi-

earlier

(3,11),

The final

con-

Added

6Data from

two separate

obtained

in nmoles

TABLE

I

M-64

were

produced

to the

per minute cultures.

used and normalized

log

phase

per urea

---

--

value.

ml of culture.

---

---

The values

13

--

225

--

--

88 114

42

are

Defective

Arginase

M-58

cehcv&k~~.

50

of Growth

---

Units

62

glyoxylate with

10 Klett

Defective

& Arginase

475

---

---

375

per

M-130 Allantoicase

-mm

66

58

66

66

Enzyme Activitya

Defective

Defective

in

Hydrolase

Allophanate

Carboxylase

Urea

M-62

SYNTHESIS IN WILD TYPE AND MUTANT STRAINS OF Sacchatramycti

Increase

experiments

curves

expressed

23gb

Arginine

are

575b

Formamide

of induction

575b

Urea

slopes

5756

Allantoate

'Activities

575

Allantoin

Type

75

Wild

M-25

RATE OF ALLANTOINASE

None

Medium

to Growth

Inducer

DIFFERENTIAL

the

z E n F

B

:

P .

Z

.!!

d.-

BIOCHEMICAL

Vol. 52, No. 1, 1973

centration

of the various

the methods

inducers

of Van de Poll,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was lo-*M.

Allantoinase

was assayed

by

the

of

& UX (12). RESULTS

Table

1 summarizes

various

compounds

the results

to serve

of experiments

as inducers

in one or more of the activities toin

will

serve

verted

to allophanic

allantoic

acid

in a mutant

This

sess a very

defective

large

arginine

lase

minus

strain

tion

of allophanate.

a hydrolase absence

of added an analogue

urea

carboxylase. to function

to carboxyformamide, ability its

synthesis.

(9)

with

allantoicase

to behave

that

the fold

lar tions.

allantoinase

sources

induction of urea

These mutations

c~,~v&&iae

are

for

effective product

of this

in Table

in wild

analogue

type

of the complex analogue.

only

and was demonstrated

similar

reported

and purines) decrease

140

are the

Forma-

of the urea

in strains

after

lack-

blocked level

and was converted for

1) to bring

its about

have been performed

These

increases

being

was tested

here

of allantoinase.

greatly

not

(Table

hydrolase.

to that

in the

to be carboxylated

Formamide

to those

of allantoinase,

induction

but

(4)

concentra-

was observed.

about

strains,

in a hydro-

of allantoinase

result

to bring

known to pos-

acid

inducer

levels

1, this

was shown

similar

amino

is the high

in an allois

allophanate

has been shown

presumably

can be con-

activity

the intracellular

of allantoinase (arginase

allan-

enzyme or its

to increase

and ureidoglycolate

in a manner

that

was observed

allantoinase

contain

as an inducer

Experiments

pear

should

an allophanate

to induce

it

compounds

this

Sacchahomycti

if

complex This

that

Degradation

As shown

of urea,

multienzyme

suggested

strain.

Therefore,

urea.

is clear

where

none of these

by monitoring

be expected

mutant

It

defective

of allantoinase.

pool.

would

defective

degradative ing

to induction

1.

instances

suggests

ability

in strains

the same behavior

that

carboxylase

was tested

hydrolase

in those

The fact

urea

is vital

in Fig.

Essentially

and urea.

hypothesis

phanate

mide,

acid.

lacking

allophanate,

only

test

of allantoinase

depicted

as an inducer

that

two enzymes It

ap-

is significant

when the

intracellu-

by appropriate

muta-

of allophanate

which

Vol.52,No.

BIOCHEMICAL

1,1973

results

in a lower

cells.

It must

allantoic

acid

differential

also is

unpublished)

rate

be pointed

caused

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

out

by urea

to contaminate

of allantoinase

all

that

the

which

synthesis

slight

induction

has been shown

commercial

in uninduced observed

(Sumrada

preparations

with

and Cooper,

of this

compound.

DISCUSSION Our results ticipating

show that

in purine

ate.

Palleroni

two enzymes

and Stanier

pathway

the

degradative

enzyme of the

procaryotic

and Schlesinger, pathway

pathway.

pathway.

in the

effect

the last trogen

intermediate it

cer

from

related the

at a minimum

results

it

available. arginine control

this

in a multi-step for

metabolites.

value.

On the other

would

It would

is

increase

presumably

be beneficial The central

and allantoin

during

element

of nitrogen

its

it

presence

of excess

intracellular

pools

levels of nitrogen nitrogenous

to function

be ni-

pools of the

indu-

remained limitation

of arginine

at the terminal

in Sacctiomycti

resides

of nitrogen

any excess

allows

intermediate

concentration

intracellular periods

in either

situation

remoteness

of

of the

an inducer

periods

of allophanate

metabolism

the

last

product

by having

its

large

these

to metabolize

catabolism

of this

In the

that

its

the

gained

situation

in the

during

position

is

assure

hand,

being

to degrade

has been shown to mobilize in a dramatic

example,

pathway.

In this

the enzymes

the first

value

an organism

of the second

acid,

is

that

that

of the first

by urocanic

of an inducer

induction

reactions

allophanate. that

upon

initial

Sacchahamycti This

However,

showed

par-

of allophan-

inducer

a product

(14)

induced

seven enzymes

presence

the

kynurenine,

The physiological

is unnecessary

of purine

that

e;t &

are

systems,

in a degradative buffering

is

of the

upon the

demonstrated

degradation

or eucaryotic

five

is contingent

(13)

of tryptophan

histidine

of at least

degradation

enzyme in the

first

synthesis

(10).

of urea

and

limitation compounds junction

as a major,

of

unique

ctievhtie.

ACKNOWLEDGMENT This work was supported by NIH grant Foundation Grant O-50 and a Brown-Hazen Lawther was supported by a Andrew Mellon

GM-19386, Health Research and Services grant-in-aid to T. G. Cooper. R. Predoctoral Fellowship award.

141

BIOCHEMICAL

Vol. 52, No. 1, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1.

DiCarlo,

F.J.,

Schultz,

A.S.,

and Kent,

A.M.,

44. 468-474 (1953). 2. :: 5. 6. 7.

Roan, R. J. and Levenberg, Whitney, P.A. and Cooper, Whitney, P.A. and Cooper, Thompson, J.F. and Muenster, 1049-1055 (1971). Roon, R.J., Hampshire, J., 7545 (1972). Whitney, P.A. and Cooper,

Arch. tiochem. tiophyd.,

B., J. Bid!. Chem., 243, 5213-5215 (1968). T.G., J. sio.L Chem., 247, 1349-1353 (1972). T.G., J. &Lob. Chem., 248, 325-330 (1973). A.E., BLochem. tiophys. Res. Cotnmun., 43, and Levenberg, T.G.,

B.,

J. BioioL.Chem., 247, 7539-

Biochem. Biophyb. Red. Conmun.., 49, 45-

51 (1972). 8. 1:: 11. 12.

Whitney, P.A., Cooper, T.G., and Magasanik, B., 1. E%oe. Chem., Submitted for publication (1973). Lawther, R. and Cooper, T.G., In preparation (1973). Whitney, P.A. and Magasanik, B., J. ti0.C. Chem., Submitted for publication (1973). Whitney, P.A. and Cooper, T.G., Biochem. tiophys. Red. Commun.,40, 814819 (1970). Van de Poll, K.W., Verwey, A.A.G., Koningsberger, V.V., P~oc. Kon. N&.

Akad. wet., ii:

Palleroni, Schlesinger,

871, 344-358 (1968).

N.J. and Stanier, S., Scotto, P., 4331-4337 (1965).

R.Y., J. GUI. Usrobioioe., 35, 319-334 (1964). and Magasanik, B., J. RLoL. Chem., 240,

142