Ribonucleotide reductase from saccharomyces cerevisiae

‘01. 41,No.

BIOCHEMICAL

1, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RIBONUCLEOTIDE REDUCTASE FROMSACCHAROMYCES CEREVISIAE* E. Vitols,

V.A. Bauer and E.C. Stanbrough

Department of Biochemistry Scripps Clinic and Research Foundation La Jolla, California 92037

Received August 21, 1970 Summary. Partially purified ribonucleotide reductase from Saccharorryces cerevisiae was unstable in solution; however, enzyme activity could be maintained by quick-freezing and+$torage at low temperatures. The reduction of CDPwas stimulated by ATP/Mg and maximal activity was obtained with E. coli thioredoxin as a reductant, bu; not with dithioerythrito1.l Enzyme activity was inhibited by dATP, hydroxyurea, and moderately high salt concentrations; addition of 5'-deoxyadenosylcobalamin or of iron salts was without effect. Gel filtration of the yeast extract yielded a fraction highly inhibitory to enzyme activity.

Ribonucleotide reductase, which in the presence of certain [R(SH)2] catalyzes

the overall

dithiols

reaction:

allosteric

effecters f Ribonucleotide f R(SH)2 cofIctors*deoxyribonucleotide

has previously

been studied in two bacterial

and LactobaciZZus leichmannii, (cf.

l-3).

species, Escherichia coZi

and in somemammaliantissues and tumors

In the present investigation

ribonucleotide

f RS2 + HZ0

yeast was selected as a source of

reductase because this organism occupies a position

between bacterial

and animal cells,

complexity of intracellular

both with regard to its

organization.

intermediate

growth rate and to its

Furthermore , yeast has an advantage

over mammaliansystems in that it can be grown under varying,

but strictly

*

This investigation was supported by a research grant (AM 12931) from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, and by Special Grant No. 439 and a DernhamSenior Fellowship in Oncology, both from the California Division of the American Cancer Society.

1.

Abbreviation

used:

DTE, dithioerythritol.

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controlled

conditions,

including

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synchronous division

Two strains of S. ceretisiae "wild"

strain

have been used in the present study:

of Fleischmann's yeast obtained commercially,

adenine auxotroph XT300-4A (4). extract-glucose-salts cooling,

of cells. a

and a leucine/

The cells were grown on a peptone-yeast

medium (5) with vigorous aeration and, after

harvested and washed by centrifugation.

rapid

A suspension (SO%, v/v)

of

packed cells in 0.05 M potassium phosphate, pH 7.5, was disrupted by ultrasonication;

the mixture was then diluted

(to reduce viscosity)

potassium phosphate, pH 7.0, and centrifuged 40,000 x g. (0.8%);

The crude cell-free

extract

protein was precipitated

with 0.025 M

twice (10 min, and 30 min) at

was treated with streptomycin

with ammonium sulfate

(0.3 g/ml),

dissolved

in 0.025 M potassium phosphate, pH 7.0, and then desalted by dialysis the samebuffer wise elution

to give Fraction

I.

Further purification

of the enzyme from a DEAEcellulose

was eluted by 0.15 M phosphate buffer, II),

contained about 2% of the total

7 uM E. coZi B thioredoxin; a total

volume of 0.4 ml.

addition of perchloric and following

was achieved by step-

then concentrated and desalted (Fraction crude extract

protein. reductase contained:

0.4 mMCDP-2-14C (1 mC/mmole); 10 mMDTE;

2.5 mMATP;

5 mMMg++ (acetate);

and 10 mMRF in

The mixture was incubated at 30' for 30 min.

acid to stop the reaction

the addition of carriers,

nucleotides were hydrolyzed

and to precipitate

the cytidine

After

protein,

and deoxycytidine

to the monophosphatesand separated on Dowex-50 (H+)

as described by I!eichard (6). measured spectrophotometrically aliquots

against

column. The material which

The standard assay mixture for ribonucleotide 10 mMpotassium phosphate, pH 7.0;

sulfate

The concentration and its

in a BeckmanLS-233 liquid

of the isolated dCMPwas

radioactivity

scintillation

was determined by counting counter.

Results are

expressed in nmoles of dCMPformed/mg protein. In our initial

experiments, ribonucleotide

be detected in crude cell-free catabolic

yeast extracts

reductase activity

could not

because of the interference

of

enzymes. Both substrate and product were degraded by deamination to

72

Vol. 41, No. 1,197O

uridine

compounds

was partially addition

and also

overcome

of fluoride

conditions, I,

BIOCHEMICAL

the

for

to inhibit

ribonucleotide

under

stages

amounts

of growth.

by harvesting

Maximal

higher

to these

cultures

thereafter. ments

be detected below

obtained

Isolation of its

24 hrs.

cation

(to

iron for

salts the

at 0’.

which

in solution,

II)

nor

by the

isolation

varying

of ribonucleotide could

by quick-freezing

be preserved

the preparation

in

from

periods liquid

from

sulfate

cell with

was added shortly

and inhibitors

could

presented

the mutant. was difficult of activity

because being

improved

less

by purifi-

EDTA, or magnesium of success other

and

as stabilizers

sources.

of up to several nitrogen

rapid

harvested

significantly

degrees

reductases for

was obtained

such as the require-

the half-life

have been used with

strain

The results

of thiols,

and

at different

cells

reductase

addition

grown

enzyme preparations

two strains.

was not

were

of yeast

when adenine

enzyme preparations

constant

to determine

of stimulators

ribonucleotide

and the specific

of most

differences

from the

and

DEAE cellulose

cells

the “wild”

and the

effects

Enzyme stability

Fraction

enzyme activity

with

instability

phase,

or the

enzymes

the

however,

no qualitative

of the yeast

extreme

than

the

that

(XT300-4A), obtained

certain

in Fraction

was relatively

two strains

from

the

enzymes,

at the stage

stationary

this,

from

(i.e.,

were

evident

was possible

in the

phase

components

between

have been

it

activity

from

of reaction

Consequently,

and by the

by determining II

fraction provided

strain

at early

Apart

this

to another,

in mid-log

specific

was still

Fraction

I)

difficulty

When, under

was calculated

enzyme activity

From the mutant

a 4-fold

in

This

of Fraction

above degradative

of enzyme present

cells

division).

the

activity

conditions.

the

activity

from

one preparation

identical

compare

free

stage

activity

of dUMP formed.

reductase

(+ 15%) from

the

bonds.

phosphorylase.

dCMP deaminase

amount

was essentially

(to

nucleotide

reductase

the

of the glycosidic

by purification

ribonucleotide

column

by breakage

some residual

correcting

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The yeast weeks,

and subsequent

however,

storage

at -20’. Under

the

optimal

assay

conditions

73

described

above,

the enzyme prepara-

Vol. 41,No.

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(both Fractions I and II)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

catalyzed

rate over a period of one hour (Fig.

the reaction

1).

Time

at a relatively

The reduction

linear

of CDPwas completely

(mid

Fig. 1. Reduction of CDPby Fractions I and II as a function of time. Standard assay conditions (see text), incubated for periods of time indicated.

dependent upon the addition

of a reducing substrate;

(chemically reduced by DTE) was a far more effective (Fig.

2).

The relation

of enzyme activity

2

Thiaedoxin 4

E.

coi!i thioredoxin

reductant

to thioredoxin

than DTE alone

concentration

(PM) 6

6

IO

I

DTE (mM)

Fig. 2. Effect of DTE and E. coZi thioredoxin concentration on CDPreduction by Fraction II. (--o--) ) activity at varying DTE concentrations (bottom scale) in the absence of thioredoxin; (--a-), activity at varying concentrations (top scale) of thioredoxin, chemically reduced in the presence of 10 n&l DTE.

74

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Vol. 41, No. 1,197O

followed simple Michaelis-Menten Previously,

E.

purified

In contrast,

kinetics,

the apparent Kmbeing about 3 PM.

the dependence upon thioredoxin

only with the recently

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for maximal activity

co7,i enzyme, for which thioredoxin,

was observed

either native or that

from yeast (7), is a better reductant than simple dithiols.

the enzymes from

sources (3,9) exhibit

L.

Zeichmmrnii (8) and from different

maximal activity

mammalian

in the presence of such dithiols

alone.

Maximal CDPreduction by the yeast enzyme depended upon the presence of ATP and Mg+', whereas low concentrations hibition

of dATP produced significant

even in the presence of 2.5 mMATP (Table I).

Addition

in-

of either

TABLE I Effect

of Reaction Componentson the Activity of Yeast Ribonucleotide Reductase

Complete System (Fraction

Activity* 7.20

II)

hission DTE

0.00

Thioredoxin Mg++

0.44 1.08

ATP

0.86

0.1 mMdATP

4.03

0.5 n&l dATP

3.67

2.5 mMHydroxyurea 5.0 mMHydroxyurea

5.04 3.89

0.05 M Phosphate, pH 7.0 0.2 M Phosphate, pH 7.0

9.36 1.44

0.2 M NaCl 0.2 M (NH4)2S04

0.77 0.00

Addition

* nmoles dCMP/mgprotein 5'-deoxyadenosylcobalamin

or iron salts did not stimulate activity,

chelating agents, such as 8-hydroxyquinoline

and orthophenanthroline

and iron (1 mM),

BIOCHEMICAL

Vol. 41, No. 1,197O

did not inhibit

activity

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when added either

directly

preincubated with the enzyme for one hour at 0".

to the assay mixtures or Preincubation

against the chelators for longer periods (10) was not feasible, instability

of the enzyme.

Activity

moderately high salt concentrations hibition

inhibited

was

(Table I).

might be due to ionic strength,

particular

ion.

Some

stimulation

or dialysis because of the

by hydroxyurea and by

It appeared that the salt in-

rather than to the presence of any

of enzyme activity

by the addition

of 0.05 M

phosphate (Table I) may have been due to the blocking of CDPdephosphorylation to the inactive inhibitory. inhibitor

A

monophosphate; however, higher phosphate concentrations

low molecular weight compoundwhich acts as a highly potent

of enzyme activity

sulfate

precipitate

was isolated by gel filtration

used to prepare Fraction

I.

pound had a spectrum reminiscent of certain liminary

ing that its molar extinction

coefficient

Thus, someof the properties E. coZi ribonucleotide

extreme instability from tumor cells

Not yet identified,

this com-

more inhibitory

than dATP, presum-

is comparable with that of nucleotides

of the yeast enzyme appear similar the requirement of thioredoxin

reductase (i.e.,

and, probably,

of the ammonium

nucleotides and appeared in pre-

experiments to be at least loo-fold

imal activity

were

possession of a tightly-bound

for max-

iron) whereas its

is more akin to the mammalianenzymes, particularly (3).

The sensitivity

to the

of the yeast ribonucleotide

those

reductase to

high ionic strength also bears someresemblance to the mammaliantumor enzymes, but

is in sharp contrast

to the bacterial

ribonucleotide

fully

active in the presence of up to 1 M concentrations

12).

It seemsunlikely

reductases which are of certain

that the rapid loss of the yeast enzyme activity

was due to contaminating proteolytic

enzymes, since the reaction

linear

and stability

at 30' for one hour (Fig.

l),

successive steps of enzyme purification. in the level of enzyme activity

(11, at 0'

rate was almost

did not improve during

Because of the observed differences

at varying stages of cell growth and because of

the probable involvement of a highly effective inhibitor,

salts

the yeast ribonucleotide

(as yet unidentified)

reductase seemslikely

76

endogenous

to provide a useful

Vol.41,No.

1, 1970

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

system for studying the manner in which the control of reductase activity be related

may

to cell division.

ACKNOWLEDGMENTS The authors are indebted to Dr. J.A. cerevisiae and helpful

strain

DeMossfor a culture

of S.

XT300-4A, and to Dr. F.M. Huennekensfor valuable comments

discussion during the course of this work.

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

Larsson, A. and Reichard, P., in Progress of NucZe?:cAcid Research and MoZecukr Biology, J.N. Davidson and W.E. Cohen, Eds., Vol. 7, Academic Press, New York, 1967, p. 303. Moore, E.C., in Methods in Enzymology, S.P. Colowick and N.O. Kaplan, Eds., Vol. XII (A), Academic Press, New York, 1967, p. 155. Blakley, R.L. and Vitols, E., Ann. Rev. Biochem. 37, 201 (1968). Manney, T.R., Duntze, W., Janosko, N. and Salazar, J., J. Bacterial. 590 (1969). Halvorson, H.O. and Spiegelman, S., J. Bacterial. 64, 207 (1952). Reichard, P., Acta Chem. Stand. 12, 2048 (1958). Porque, P.G., Baldesten, A. and Reichard, P., J. Biol. (1970).

99,

Chem. 245, 2363

Vitols, E. and Blakley, R.L., Biochem. Biophys. Res. Commun.2, 466 (1965). Larsson, A., European J. Biochem. c, 113 (1969). Brown, N.C., Eliasson, R. Reichard, P. and Thelander, L., Biochem. Biophys. Res. Commun.30, 522 (1968).

11.

Brown, N.C., Canellakis, Z.N., Lundin, B., Reichard, P. and Thelander, L., European J. Biochem. 2, 561 (1969).

12.

Jacobsen, D.W. and Huennekens, F.M., Biochem. Biophys. Res. Commun.37, 793 (1969).

77