Vol.

No.

177,

June

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

14, 1991

SPECIFICITY

OF GUANYLIC RNASES TO POLYNUCLEOTIDE SUBSTRATES

Valentin

'Institute

630-635

BothI.

Gennady

P.Moiseyev2

and Jozef

of Molecular Biology of the Slovak Dubravska cesta 21, 842 51 Bratislava.

'Engelhardt

Institute of Molecular Sciences, Vavilova

Received April

12, 1991

Sevcik’

Academy of Sciences, Czechoslovakia

Biology of the USSR Academy of 32, Moscow, USSR

Kinetic parameters &cat and KM were measured for cleavage of poly I. poly A, poly U, poly C and poly 1.~01~ C by guanylspecific RNases Sa Pbl and Ti and compared with that of guanylefficiencies of the investigated preferential RNase Bi. Catalytic enzymes to polynucleotide substrates vary considerably. The structural basis for specificity of these RNases is discussed. A hypothesis is suggested that Ser-56 plays an important role in recognition of poly A by RNase Bi. 0 1991Academic Press,Inc. Up to now nucleases

dealt

with

oligonucleotides

on the

(l-6).

RNases is

However,

to degrade

serve

as models

tions

(7-8)

long

of

these

dealing

with

guanyl-specific

specificity

interactions

as substrates

as inhibitors of

papers

most

of

these

or with one of

ribo-

enzymes with

G-containing

short

nucleotides

the main physiological

RNA chains.

substrates.

There

no complete

roles

Polynucleotides

depolymerization

RNases but

of guanylic

are only

could

few publica-

of polynucleotides kinetic

parameters

by (kcat,

KM) are presented. Beside described only

which,

guanylic

specificity is

guanyl-specific

guanyl-preferential

and GpN but

in polymeric

substrates

necessary

to characterize

of

these

RNases are

low-molecular-weight-substrates,

among

cyclophosphate

therefore

specificity

RNases.

RNases using

loose

hydrolyze their

(RNases Bi,and quantitatively

different

guanylic Ba)

(9-10).

It

the

polynucleotides

as

substrates. MATERIALS AND METHODS RNase Sa from purified according

Streptomyces aureofaciens to (11). A homogeneous

0006-291X/91$1.50 Copyright All rights

0 1991 by Academic Press. Inc. of reproduction in any form reserved.

630

was isolated and sample of RNase Pbl

Vol.

177,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

from Pacillium brevicompactum was isolated according to (12) and generously supplied to us by Dr A.L.Bocharov (Inst.Mol. Biol., Moscow). RNase Bi from Bacillus intermedius 7P was kindly supplied by Dr N.K. Chepurnova (Inst.Mol.Biol..Moscow) and RNase Timfrom hernillus orysae from Sankyo (Tokyo; Japan) was used without further purification. Poly I, poly A, poly U and poly C were from Reanal (Budapest, Hungary) and were purified to remove shorter chains as described in (13). G>p (guanosine-2’:3’cyclophosphate) was purchased from Sigma. The depolymerization reactions were carried out in Tris acetate buffer, pH 6.2 in thermostatted spectrophotometric cells at 25kO.05 OC. The initial concentrations of polynucleotides were determined spectrophotometrically using molar extinction coefficients according to (14-15). The initial reaction rate was determined as the increase of optical density in time, using difference molar extinction coefficients of polynucleotide conversion according to (13). For spectrophotometric measurements a Specord M-40 (Karl Zeiss, GDR) was used. Kinetic measurements of kcat and KM for G>p were carried out on a pH-stat (Radiometer, Denmark) as described in (16). At different concentrations for each substrate, 6-8 initial rates were measured and the kinetic parameters were calculated from Lineweaver-Burk plots (17) using the least square method.

RESULTS Guanylic gation.

were

of

the

rate

for

poly

I

kinetic

cleave

This

is

why

data

RNA in

the of

of

by different good

in

Bi

from

(IO).

two

guanylic agreement

the

has

on the

are

for

yield

631

analog specificity 7P,

comparison.

These

G>p were

practically close

(Pbl, features.

interactions

RNase rates

fungal

common

site

for

RNases.

of many

has

complexes of

(18).

G>p cleavage

identical, homology

in-

3'-phosphate

measured of

examined the

as

a terminal

revealed

rather

subst-

intermedius

structures

that

a ten-

structural

efficiencies

I and 2 show

with

G (which

2' .3'-cyclophosphate

for

in

RNases

specificity

an unsuitable

active

identical

guanylic

shown

to

RNases

investi-

polynucleotides

Bacillus

with

nucleotide-protein

Table

kinetic

2 data

are

three-dimensional Bi)

the

closest

Table

catalytic

RNases

being

from

steps

this

poly

its

parameters

(Sa.

to be nearly

Data

in

of

RNase

kinetic

and bacterial

proven

thus

In

for

the

of

by hydrolysis

relative

Comparison

The pattern

instead

as

chosen

different

comparing

substrate.

followed

comparison

is

as

with

termediate

of

measurements)

used

of

solution

guanyl-preferential

enzymes

For

RNases in

were

specificity

1).

aggregate

was

and Ti

reactions

(Table

supplemented

Tl)

the

investigated to

Pbl

cleavage

measured

dency

of

Sa,

To determine

parameters

of

RNases

AND DISCUSSION

which of

their

Vol.

177,

No.

2, 1991

BIOCHEMICAL

AND

Substrate

Tl

COMMUNICATIONS

I

kcat

CM)

(l/s)

K”Xi04

kcat/KM

CM)

(l/s.M)

1.33

2.9X106

POlY I

0.1

385

POSY poly

A C

0.1

1. 5x1o-3

25

0.6

0.1

b

b


P

1 A C D 1.pol.y

c"

0.25

485

ca

8.33

5.8~10~

1x1o-3

20

0.25

b

b


P

in Table

three

I hydrolysis,

a factor

It

with

are not

be seyara-

not

guanylic cleaved

1 indicate

RNases are rather the

four

that

mononucleoby these

RNases

RNases Sa, Pbl

One can see,

and

kcat

and six,

U and poly A cleavage

KM

is

that

that

the

between

and equals small,

but

of RNases Sa and Tl on poly

of guanyl-specific

RNases. As a probable 632

instance

by not

None of

ratio

and TL

kinetic For

differ

respectively.

identical

the

similar.

values

C and the

can be presumed

activity

curve.

shown).

of all

RNases split poly

and

guanyl-specific.

parameters in poly

progress

KM

in complexes

U>p,

not

the

value kcat and KM could from the Lineveawer-Burk plot.

presented

Data

of

structures

(18).

at all

tangent the high

more

these the

rates

to about

of

10"

definitely

A is

an intrinsic

explanation

of

Vol.

177,

No.

2, 1991

BIOCHEMICAL

Kinetic

AND

I

POIY I d POIY A POIY u poly Cd

I.poly

(l/s)

0.2 0.2 0.2 0.2 0.2 0.2

c

G>P

of

Knx 1 o4

kcat

(M)

poly

COMMUNICATIONS

reaction G>p by guanyl-preferential Bi at PH 6.2

and RNase

RNase Substrate Bi

RESEARCH

Table 2 uf the cleavage

parameters

polynucleotides

BIOPHYSICAL

kcat/Ku

CM)

55 77 2 0.025 c 0.1

(l/s.M)

0.22 4.0 50 1.7 c 1.7

3.5w106 1. 9x106 400 1.5x102 4.0x104 5.9x10"

c) See footnote c in Table 1. d) Data taken from (101

this

phenomenon the

guanosine

closer

as compared

However,

should

structural

to uridine

this

be the case,

and poly

be much different

lo-*

the

i\l-ls-l

The rates within

its

to

double that

mation

of

It

(IO),

polyphosphate cally

with

what causes higher earlier

(Table

stranded

(10)

fragment 2 that

the

charged

the main

appear

have

highest

groups

a low kcat

not

U and poly reason

633

for

the

in

(as compared the confor-

optimal

RNase Bi

(13).

loses

its

guanylic

As was suggested

phenomenon interacts at

rate

spontaneously

because

is

this

A to be cleaved poly

RNases vary

by RNases because

substrates.

substrate

that

I

to be

cleaved

polynucleotide)

Table

of

than

G>p and A>p.

are

backbone

efficiency

of

As was shown earlier,

for

poly

of poly

I).

the main reason

the

by

ratios

by guanylic

which

polynucleotide

positively

hydrolyzed

A>p can be expected

These enzymes

can be seen from

those

be mentioned.

and RNase Sa has the

the phosphodiester

for

from

C cleavage

nucleotides

(13).

single

specificity earlier

1.~01~

polynucleotides

he1 ix

rate

to

low to be measured.

lowest

exposed

for

cleavage

for

of magnitude

KM is

enzyme binds

too

of poly

double-stranded

the

is

one order

because

value

kcat/KM

which

the

adenosine

could

why is A>p not

Possibly,

Consequently,

of

or cytidine

RNases Sa and Tl? A may not

similarity

the

is

that

electrostati-

protein

surface.

by RNase Bi with C ?. this

It

the

a much

was suggested

phenomenon

is

the

But

BIOCHEMICALAND

Vol. 177, No. 2, 1991 better

stacking

residue

in

account

recent

bond

between

side

chain

could

the

active

the

3.0

8 and

is

2.5

8.

This by

poly

and

virasole>p bond

in

with

nucleotide

has

RNases

N7 protonation

in

and

in

Besides,

the

chemical

change Possible

mutagenesis of

Ser-56

Bi-nucleotide

ll'N1l

ways to (which

to

obtain

of

complex

at

shift 3'GMP check to

solved

Pbl the

Bi

or "N

attempt by 2-D

the

Pbl

and

Sa)

of

RNase

Bi

of

is

Ser-56 and N7

suppor-

purineposition poly

as

A and and

Tl,

of

Pbl

and

the

nucleotide Tl

(5)

in and

Sa (20).

NMR spectrum RNase

Tl

does (19).

be site-directed

containing

substitutions

NMR in

or

structure

Sa

pK of

would

soon)

Bi.

Non-existence

with

hypothesis mutants

of

pK values: the

complexation our

RNase

for

N7 in

atom

l-Me-Ino>p

the

RNases

of

equally

RNases

identical with

is

(161.

by measuring is

RNase

rates

do not

hydrogen

of Y NH Ser-56

of

cleaves

into

N7 and 0

an analogous

guanyl-specific

shown

we hope

Bi

)I

Tl,

chain

relative

and Pbl

complexes

upon

residue

RNase

Tl

3'GMP

solution

not

b/

main

Ba (which

similar

complexes was

RNase

and 0

aromatic

taking

the

preference

preference

a Ser

(IO);

but

such

a/

that

between the

the

and

RNases

purine

distance

purine

exhibits

I cleavage

the

the

now

in

with

this,

base

absent

between

that also

Bi

for

of

base to

guanine

is

distance

fact,

adenine

we presume

the

(which

data,

the

preferential) RNase

of

atom

explanation the

(19)

responsible

to X-ray

is

in

N7

the

In addition

data

Ser-56

be also

of

site.

X-ray

of

According

ted

interaction

BIOPHYSICALRESEARCH COMMUNICATIONS

of

RNase

solution.

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AND

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R.W.,

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