Vol. 64, No. 1, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

EVIDENCE FOR METALLOENZYME CHARACTER OF tRiiA hlJCLEOTIDYL TP,AivSFERASE Kenneth

R. Williams

and Peter

Schofield

Department of Biochemistry University of Vermont Burlington, Vermont Received

March

18,1975

SUMMARY Previous studies (1-S) have shown that several nucleotidyl transferases are metalloenzymes and in a few cases (l-3) the metal nas been identified as zinc. In all instances these enzymes are specifically inhibited by incubation with the chelating agent l,lO-phenanthroline but are not affected by the structurally similar 1,7-phenanthroline which does not chelate metals. We report here that tRNA nucleotidyl transferase from E. coli is inhibited by l,lO-phenanthroline and that only the initial rate of incorporation of AMP is affected; CMP incorporation This finding is in conflict with is not specifically inhibited by this chelator. a previous study (5) in which it was claimed that tRNA nucleotidyl transferase from Rous sarcoma virus and from yeast was unaffected by l,lO-phenanthroline and transferase is a metalloenzyme. suggests that the E. coli tRNA nucleotidyl

Several

nucleotidyl

instances,

the metal

that

the

zinc

per molecule

transferases has been

DNA-dependent

of enzyme while

and sea urchin

per molecule

of enzyme.

are

from avian

specifically

transferase

from

calf

and sea urchin

virus

(5).

the metal

ion

in terminal

of the deoxynucleotide

Copyright All rights

(S),

On the basis

these

various

from E. coli

contains

two atoms of bound

et al

showed

(2)

Auld

(3)

recently,

et al

gland

and the of kinetic

In addition,

the

the

terminal

data,

nucleotidyl

o 19 75 bv Academic Press, Inc. of reproduction in any form reserved.

DNA polymerase

transferase

transferases 262

the

following

is

(4)

deoxynucleotidyl

from

from rat Rous sarcoma

postulated

required

in the reaction and the

DNA enzymes

RNA polymerase

Chang and Bollum

The similarities

of zinc

mole of the RNA-dependent

DNA dependent

RNA-dependent

DNA polymerases

have demonstrated

virus.

(4),

the

(1) reported

respectively

per

deoxynucleotidyl substrate.

that

of zinc

by l,lO-phenanthroline:

thymus

in a few

et al

molecules

myeloblastosis

and,

Scrutton

two and four

two moles

inhibited

liver

between

More

to be metalloenzymes

as zinc.

Slater

contain

of approximately

polymerase

identified

RNA polymerase

from E. coli

existence

appear

for

that the binding

catalyzed

tRNA adenyl(cytidyl)trans-

BIOCHEMICAL

Vol. 64, No. 1, 1975

ferase

(E.C.

heretofore

2.7 . 7.25) undetected

Materials

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

led us to analyze

metal

is

to be published

enzyme used

in these

optimum

reported

studies

a specific

the presence

purified

preparations.

of a

and 0.2

pli 9.0,

and washed

with

and counted and only

acid.

the average

Frederick

Smith

Company.

The Z,8-(3H)

Results

based

was stopped

In all

than

The 1,7-phenanthroline

l,lO-phenanthroline

ATP and s-(~H) CTP were

were

filters

the

were

dried

done in triplicate average

deviation was from

was from obtained

fiber

The filters

assays cases

or L?-(~H)CTP

of 5.0 ml of cold

on glass

reported.

Company and the

of g-mercaptoethanol,

by the addition

and 95% ethanol.

highly of 0.1 ml:

of 2,8-(3H)ATP

was collected

10%.

their

volume

1.0 umole nCi)

a specific

for

in a final

All

was less

tRNA was from

of protein

(0.25

of protein

(9) claim

scintillator.

are

of protein

the Aldrich

from New England

of the G.

Chemical Nuclear

Schwarz-Mann.

and Discussion

Initial

studies

I,lO-phenanthroline 2 mM B-mercaptoethanol activity was added observed.

et al

of MgC12,

nanomoles

acid

of the purified

(8) have previously

contained

The precipitate

values

assays

the yeast

40.0

5% trichloroacetic

in a toluene

multiple

1.0 pmole

The reaction

10% trichloroacetic

these

tRNA,

ug of protein.

Carre

assay

B by a method

AMP incorporated/min/mg

AMP incorporated/min/mg

The standard

of yeast

activity

and Philipps

while

E. coli

AMP incorporated/min./mg

of 480 nanomoles

nanomoles

of Tris-Xl,

2.0 nanomoles

Miller

preparation,

from

specific

was 485 nanomoles

at 37°C.

homogeneous

of 1,000

5.0 nmoles

The final

activity

activity

was purified

(10).

conditions

a nearly

while

for

and Methods

which

for

latter

ion.

The tRNA adenyl(cytidyl)transferase

under

the

as measured directly In view

demonstrated

that

in the presence

preincubation of 10 mM Tris-HCl,

and 10% glycerol

led

by AMP incorporation. to the of this

standard discrepancy

of the

assay

pH 6.8,

to a loss However,

system

enzyme with

of nearly

10 mM MgCl, 40% of the enzyme

when l,lO-phenanthroline

no significant

each of the

0.1 mM

components

inhibition of the

was assay

system

BIOCHEMICAL

Vol. 64, No. 1,1975

was examined

to determine

its

effect

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on the

extent

of inhibition

by l,lO-phenan-

throline. As can be seen in table in a five inhibition

minute is

incubation

independent

in the presence

However,

then

70% of the

reported found

if

observed

the

of ATP.

If the

inclusion with

tRNA,

then

original

activity

et al

(5)

of 14.3

I. EFFECT

*RNA-

NUCLEOTIDYL

incubation

is

9.0

pH

9.0,

pH

6.0

is

incubation

done at pH 6.8

remains

RNA polymerase

or pH 9.0 or at

after

even in the absence

A similar II

phenomenon

from rat

decreased

lost

This

is done on ice,

included,

remains.

liver.

five of tRNA,

has been They

the inhibition

by 49%.

OF

ASSAY

COMPONENTS

TRANSFERASE

CONDITIONS

pH

is

mM l,lO-phenanthroline.

mM S-mercaptoethanol

l,lO-phenanthroline

TABLE

for

activity

30 % of the activity

B-mercaptoethanol

by Valenzuela that

0.25

of whether

of yeast

minutes.

90 % of the AMP incorporating

at 37OC with

or absence

370C in the presence

over

1, nearly

BY

ON I,10

INHIBITION

PHENAN

OF

TH ROLI NE

% INHIBITION

92 0’

69 87

+ 0.4

mM

+ 0.5

mg/ml

+I0

mM

ATP

91 tRNA

70

ESH

30

Table 1: All incubation mixtures contained 50 mM Tris-HCl, 10 mM MgC12, and 0.25 mM 1, lo-phenanthroline. Other additions are as indicated above. After incubation for 5 min. , and at 370, pH 9.0 unless otherwise indicated, samples were withdrawn and assayed in the standard system minus B-mercaptoethanol (ESH). Control samples without l,lO-phenanthroline were run for each set of conditions and the % inhibition was determined by comparison to these controls.

264

BIOCHEMICAL

Vol. 64, No. 1,1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

50

50

-

25

-

CMP

02

15

4 PHE

60

250

NANTHROLIN

E

I.000 (uM)

Fig. 1 Inhibition by phenanthroline. Samples were assayed either for AMP incorporation (top graph) or for CMP incorporation (lower graph) using the standard assay without &mercaptoethanol. In addition, all assays contained the indicated concentrations of phenanthroline and 2% methanol. O-O 1,7-phenanthroline; X-X l-lo-phenanthroline.

The effect of inhibition

was further

by l,lO-phenanthroline

incorporation.

As shown

is reached

at about

10 UM reported (5) * activity

studied

Under is

for the lost;

in figure

by investigating and by assaying 1, maximal

4-8 UM l,lO-phenanthroline maximal

conditions however,

inhibition citedin

the concentration

inhibition which

of Rous sarcoma figure

more extensive

CMP as well

as AMP

of AMP incorporation is

similar

virus

to the value

reverse

transcriptase

1, 50% of the AMP incorporating inhibition

265

for

dependence

is

observed

by preincubation

of

BIOCHEMICAL

Vol. 64, No. 1,1975

of the enzyme with achieve

l,lO-phenanthroline

the same level

phenanthroline if

phenanthroline analogue

the

series

to obtain

of CMP activity

inhibition

is probably

for

the It

et al

with

(11)

DNA-dependent has long

with

for

for

reported

that

the

purified

fractions

maximal

even though

Mgtt

Chapeville

(9) have since dissociates

the inhibition

that

due to Mg++ chelation the ph is

1.5

because

(12),

and also

enzyme to l,lO-phenanthroline, inhibition metal

of AMP incorporation chelation

significant

effect

since

required

present

absence

we observe

chelating

same extent

of

and this phenan-

has been reported

and by Scrutton

et al

its

Mg ++-free

in the assay

of Mg++ or on the

stability,

(1)

buffers

lost

mixture.

instances

has been present

observed

the non-chelating

where

all

Carre

on the AMP incorporating

activity

of EDTA.

However, cannot

chelator

for

266

1,7-phenanthroline it

be

Mg++,

we have exposed

in excess.

unless

activity and

the

The maximal

at 4-8 uM l,lO-phenanthroline analogue

(8)

transferase

addition

of this

mM

since

of l,lO-phenanthroline

low affinity in all

s-10

and Philipps

the tRNA - tRNA nucleotidyl

of the

Mgtt

requires

Miller

Mg++ for

in the presence

because

transferase

In addition,

against

shown that

in the

by

of the aromatic

dehydrogenase

(7).

dialyzed

was subsequently

metal

phenomenon

tRNA nucleotidyl

activity

were

A similar

required

concentration

interaction

of

from E. coli.

enzyme from E. coli that

exhibits in excess

show the

phenanthroline

alcohol

RNA polymerase

been known that

MgC12 in vitro

complex

yeast

the non-chelating

of its

and 1,7-phenanthroline

the enzyme.

of l,lO-

of AMP incorporation

on the basis

varying

ability

at concentrations

inhibition

l,lO-

of magnitude

of l,lO-phenanthroline

due to a non-specific

system

by Anderson

1,lO

chelating

to

the

1, 1,7-phenanthroline

concentration

be explained

In order

two orders

was done using

only

Hence the

both

inhibition

ring

20X the

can best

In contrast,

throline

of experiments

inhibition.

l,lO-phenanthroline ability.

by at least

of AMP incorporation

at least

maximal

be increased

1.

of CMP incorporation,

As seen in figure

inhibition is

rate

was due to the metal

1,7-phenanthroline.

0.2 mM which

of the

must

inhibition

a similar

significant

as was shown in table

of inhibition

concentration

To determine

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

due to

has no is present

in at

BIOCHEMICAL

Vol. 64, No. 1, 1975

least

twenty

because

times

dilution

increase metal

Recently

Carre

reagents

inhibit

of CMP into sulfhydryl separate

ion,

binding

to identify

maximal

that

only

kinetics

to give

of AXP incorporation.

reported

that

on the is

We are

inhibition

a

blocking incorporation

sensitive

supports

ATP and CTP.

involved

sulfhydryl

effect

strongly

metal

enzyme fails

rates

of l,lO-phenanthroline transition

reversible

the enzyme requires

AMP incorporation

on the enzyme for

readily

that

but are without

and l,lO-phenanthroline

the particular

in tRNA nucleotidyl

for

treated

appears

(6) have also

The fact

sites the

It

zinc,

AYP incorporation

reagents

investigating

in activity.

and Cnapeville

tRNA.

is not

of l,lO-phenanthroline

possibly

RESEARCH COMMUNICATIONS

The inhibition

concentration.

or dialysis

any significant transition

this

AND BIOPHYSICAL

to both

the notion

of

currently and are

and determine

attempting its

function

transferase.

ACKNOWLEDGEMENTS This We would

work like

was supported to thank John

by a grant from Donaghy for his

the National Institutes of Health. excellent technical assistance.

REFERENCES

2. 3. 4. 5.

6. 7. 8. 9.

10. 11. 12.

Scrutton, M. C., Wu, C. W. and Goldthwait, D. A. (1971), Proc. Nat. Acad. Sci. USA 68, 2497. Slater, J. P.>blildvan, A. S. and Loeb, L. A. (1971), Biochem. Biophys. Res. Comm. 44, 37. Auld, D. S., Kawaguchi, II., Livingston, D. M. and Vallee, B. L. (1974), Biochem. Biophys. Res. Comm. 57, 967. Chang, L. M. and Bollum, F. J. (1970), Proc. Nat. Acad. Sci. USA 65, 1041. Valenzuela, P., Morris, R. W., Faras, A., Levinson, W. and Rutter, W. J. (1973), Biochem. Biophys. Res. Comm. x, 1036. Carre, D. S., and Chapeville, F. (1974), Biochim. Biophys. Acta 361, 176. Daniel, V. and Littauer, V. Z. (1963), J. Biol. Chem. 238, 2102. Miller, J. P. and Philipps, G. R. (1971), J. Biol. Chem. 246, 1274. Carre, D. S., Litvac, S. and Chapeville, F. (1974), Biochim. Biophys. Acta 361, 185. Williams, K. R. and Schofield, P., manuscript in preparation. Anderson, B. M., Reynolds, M. L. and Anderson, C. D. (1966), Biochim. Biophys. Acta 113, 235. Sane, K., Krumholz, P. and Stammreich, Ii. (1955), J. Amer. Chem. Sot. 77, 777.

267