Vol. 74, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL

THE ROLE OF GTP IN THE ACTIVATION

RESEARCH COMMUNICATIONS

OF ADENYLATE CYCLASE

by Alexander Department

of Biological

Levitzki

Chemistry,

The Hebrew

Jerusalem, Received

December

of Jerusalem

University

Israel

21,1976

SUMMARY An attempt is made to integrate the knowledge on the role of hormones and guanyl nucleotides in regulating adenylate cyclase into a single molecuthat the hormone catalyzes the activation of lar model . It is suggested the enzyme adenylate cyclase by facilitating the conversion of the enzyme from its inactive state to its active form. The hormone is also responsible for the termination of the signal namely the deactivation of the enzyme by inducing the hydrolysis of GTP at its regulatory site. The relative rates of these two processes determine the steady state concentration of the active form of the enzyme. The model also explains the difference in behaviour between GTP and its non-hydrolyzable analogs GppNHp and GTPyS. GTP was found dependent

to stimulate

adenylate

cyclases

as an intracellular regulatory the that

hormone-dependent the presence

turkey of

stable

general

Abbreviations:

the

possess model

is

interaction

and activates found

s-receptor

blocked

to study and the

the guanyl

the

it

active

1154

true

adenylate

the

and in

and an

was discovered

dependent

that

GTPase incapable

by GTPyS (5). cyclase

interrelationships nucleotide

enzyme with

fashion

regulatory

Avian

seem to offer

between

GTP, guanosine triphosphate; GppNHp, guanylyl GTPyS, guanosine-5'-0(3-thiotriphosphate). phosphate;

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

a specific

and GTPvS activate

irreversibly

dependent

GTP functions

to be generally

of a highly

More recently

B-receptor

system

with

therein)

formation

a specific

GppNHp and which

that

in a quasi-irreversible

cyclase.

possess

which

hormone-receptor

induce

adenylate

erythrocytes

erythrocytes a useful

GppNHp (3 and references

of hormone

agreed

It was also

cyclase

of hormone-

interacts

system

manner.

adenylate

hydrolyzing

which

on the receptor-cyclase

the GTP analogs

extremely

activator

in a synergistic

of a variety

It is generally

(l-4).

allosteric

site

hormone

the activation

sites. imidodi-

the

BIOCHEMICAL

Vol. 74, No. 3, 1977

This

statement

is

The adenylate negligible

justified

cyclase basal

on the

activity

activity

using

direct

binding

of a specific detailed

B-receptor

studies

outlined cyclases

properties

outlined

direct

are

evidence

only

in turkey

above

the known

facts

within

(d) a

hormone

Although

in a number

and

some of the

of hormone-dependent

erythrocytes

the frame

existence

of a specific

(5).

Therefore

the

(11-12);

existence

and pigeon

recognized.

for

between

the

recognized

or fluoride

characterized

has been provided

and (e)

(a)

possesses

by B-agonists

GTPase has been demonstrated above

adenylate

to integrate

(13-14)

findings:

membranes

are well

(c)

subunit

following

erythrocyte

cells

(6-10);

RESEARCH COMMUNICATIONS

on the interrelationship

performed

dependent

features

on these

analysis

GppNHp has been

of the

in avian

GTP regulatory

kinetic

basis

and can be activated

(b) the B-receptors

ions;

AND BIOPHYSICAL

are

an attempt

all

the

will

of a single

be made

molecular

model.

RESULTS AND DISCUSSION Basic

Postulates (1)

of the Model

Binding

presence

hormone

to the

receptor

is

independent

basis

of the

of the

of GTP or GTP analogs. This

postulate

3H-propanolol well

of the

binding

as agonist

system

is justified (9)

binding

The binding

supported

by the

to the B-receptor

finding

state

GppNHp,

thoroughly

B-agonist (3) coupled

then

(14).

that

when the

Only

to a specific

the

in

whether

by GppNHp agonist

or not.

red

andonly

cell

which

coupled

for

This these

cyclase

This

are

to adenylate

statements

(5). 1155

postulate

has been

exposed

activated. is

also

of hydrolyzing recently

is to its

to the

cyclase

incapable

as

the

first

exposed

the enzyme becomes

GTPase is

(15)

can be activated

membranes

subsequently

step

is

independent.

adenylate

that

binding

identical

the last

GTPase.

Evidence

are

hormone

turkey

washed

The B-receptor

GppNHp or GTPYS. presented

activated

of GppNHp is

super-active

observation

and 1251-hydroxybenzylpindolol

has been previously (2)

on the

to

Vol.74,No.

BIOCHEMICAL

3,1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

The Model The activation and the

of adenylate

termination

of the

Ml (1)

cyclase

hormone

by hormone

signal

can be represented

k3

R.E*GTP 2 H.R.E.GTP

and guanyl

nucleotides

as follows:

k4

-t

H*R.E'.GTP

+

H*R'E

+ GDP + P;

k2 where the

R is the enzyme.

R remains the

In the

receptor,

course

unchanged;

rate

constant

inactive

form

species

HRE is

the

total

the

active

(2)

hormone

of enzyme

the

process

k3 whereas

E is

E the

due to the

of enzyme

for it

H.R.E'.GTP

is

are

the maximal

given

enzyme

that

by

E' to its

GTP molecule.

the

of

receptor

characterized

active

bound

form

of the

of activation.

can be shown

The

If[E,]denotes

concentration

of

by: .

kl

[H]

k3

given

in the Appendix.

concentration

of HR.E'.GTP _

[H*R.E'.GTP]max

(3)

is

=

klk3 details

activation

cycle

k2k4 k4 k4 + - + (1 + -) More

nature

of the

another

activated

the

of the

hydrolysis

enzyme concentration

(H.R.E'.GTP)

activation

the conversion

now available

species

enzyme and E' the

At very

high

attainable t-L]

hormone

is given

concentrations

by

.

1 •t k4

The values

of k4 and k3 have been measured

by substituting

GTP with

characterized the

rate

these

by the

rate

of accumulation

constant

thus

eliminating

k4.

Under

of H.R.E'.GppNHp

k3 can be measured

the hydrolytic these

conditions

and k4 = 0.

step one follows

Therefore,

under

conditions: [H.R.E'.G~~NH~]

(4) namely, found

GppNHp,

independently.

all for

the enzyme kg is

0.7 min

is -1

= [E,]

converted for

to its

adenylate

1156

highly

cyclase

active in turkey

form

E’.

erythrocyte

The value

Vol. 74, No. 3, 1977

membranes

(14).

Selinger 6.0

for

min-'

that

BIOCHEMICAL

The value turkey

(15).

erythrocyte

membranes

the values

steady

state

follows

therefore

the enzyme

is

concentrat clear

that

present

ion when

that

in the

the natura

7 to 10 more active the

permanently

reverted

than

active

to the (14).

model.

GTP replaces

thus

converting

of hormone fact

that

ATP is

ATP is

since

the PM (16)

produce

the

since

of the

highly

activated

[Ed]

state

of

It

with

a contamination

ATP)

hydrolyzed

that

ATP is effective

enough

the enzyme

inactive

at the

of GppNHp can be presence

of to this

regulatory in the

site

presence

form may be due to the

GTP to produce

the reversal

of 1 x 10m4 M to produce to the guanyl

is

shown that

according

then

enzyme

is therefore

recently

in the

be explained

total

nucleotide

of 0.1% GTP in the

ATP is

effect.

the effect is

and

in the

sufficient

presented

has been analyzed adenylate

cyclase

in a quantitative but may be of general

of TTP and GppNHp described adenylate

fashion

to

common to most

text

one can write:

cyclases.

the

scheme of equation

=[R..E.GTP]

+[H.R.E.GTP]

(1)

of the

+[H..R.E'.GTP]

1157

for

significance,

are

APPENDIX

(1)

is

active

in the presence

GTP (or

enzyme to its

of the GTP site

erythrocyte

Using

enzyme

effect.

the effects

hormone

The fact

contaminated

range

super-active

It was also

can also

used at concentrations

The model the turkey

using

GppNHp and is

affinity

of the

GTP is present.

of the enzyme

phenomenon

the

to

one can conclude

of 12% to 15% of the

absence.

state

E' to E.

and

to be k3 = 4.0 (3)

of GppNHp or GTPyS and hormone

state

in reverting

Since

0.1

This

extent

1 effector

in its

inactive

hormone

concentration

to the

presence

of k3 and K4 into

by Cassel

EC! = 2 to . 645 a.5

the

only

independently

and was found

concentration

[H.R.E'.GTP]

(5) It

of k4 was measured

Inserting

the maximal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

if

not

all

BIOCHEMICAL

Vol. 74, No. 3, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

but (2)

[H*R.E.GTP](k2+k3)

= k,[R.E.GTP][H]

namely (3)

kl = k2+k3

[H.R.E.GTP]

Also (4.)

k3[H.R.E.GTPg

[R*E.GTP][H]

.

= k4[H.R.E'.GTP]

namely (5)

CH.R.EI .~TP]

i ntroduci (6)

ng (3)

[H.R.E'

(7)

[EJ

into

.GTP] (3)

Introducing

k3 = ~H.R.E.GTP]; 4 (5)

one obtains:

k3 = G x and ($)

kl k,ik, into

[R.E.GTp][H]. (i)

one obtains: k3 kl +x -[R.E.GTP][H].

kl

=[R.E.GTPI = -[R.E.GTP][H]

k4

k2+k3 (8)

[R.E.GTP]

CL1

= 1+

k +3,

kl [HI k2+k3

Introducing

(8)

into

(6)

CL1 k [HI l

[li.R.E'.GTP]

=

=

kl

[HI (1 + ---I k2+k3

one obtains: CEolCHl

4 1+ k1

CHl(l

k2+k3

k3 + ---I k4

namely: (10)

CHS~S~'U~~p]-

CLICHI k2+k3 T

k4 k4 k3 x kg + q (1 + @HI

CL1 [HI (11)

[H.R*E'.GTP]

=

k

k 2k4 t .-it k + (1 ++[H] 55 kl k3

1158

. k3

l+-

k4 k2+k3

k3 r x + (9)

k2+k3

.

k4

BIOCHEMICAL

Vol. 74, No. 3, 1977

At saturating

(12)

[H.R*E'.GTP]

hormone

concentrations

= kq 1+T;-

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

equation

(11)

is

transformed

into

. 3

REFERENCES 1. RcIdbell. M.. Birnbaumer, L., Pohl, S.L. and Kraus, H.M.J. (1971a) J.Biol.Chel n.. 246, 1877-1882. 2. Rodbell, M., Kraus, H.M.J., Pohl, S.L. and Birnbaumer, L. (1971b) J.Biol.Chem. 246, 1872-1876. 3. Londos, C., Sfimon, Y., Lin, M.C.,Harwood, J.P., Schramm, M., Wolff, J and Rodbell, M. (1974) Prl oc.Nat.Acad.Sci. 71, 3087-3091. 4. S&amn, M., and Rodbell, Mm 2 J.:;;;.;;IL 290, 2232-2236. Z. (1976) BG Fhyacta (in press). Cassel, D., and singer, 65: Levitzki, A., Atlas, D., and Steer,-1974)- oc.Nat.Acad.Sci. 71, 2773-2776. 7. zrbach, D.G., Fedak, S.A., Woodward, C.J., Polmer, J.S., Hauser, D., and Troxler, F. (1974) Science 186, 1223-1224. 8. Atlas, D., Steer, M.L., andvitzki, A. (1974) Proc.Nat.Acad.Sci.

7J, 4246-4248. 9. Levitzki, A., Sevilla, 10. i:: 143: 15. 16.

N., Atlas, D., and Steer, M.L. (1975) J.Mol. Biol. 97, 35-53. Brown, E.M., Rodbard, D.. Fedak, S.A.. Woodward. C.J.. and Aurbach. G.D. (1976)-J.Biol.Chem.-251, 1239-1246. . . Pfeuffer, T. and He1mreicrE.J.M. (1975) J.Biol.Chem. 250, 867-875. Pfeuffer, T., and Helmreich, E.J.M. (1976) J.Biol.Chem.Tn press). Levitzki, A., Sevilla, N., Steer, M.L. (1976) J.Supramolec.Str. 4, 405-4 Sevilla, N., Steer, M.L., and Levitzki, A. (1976) Biochemistr (in press Spiegel, A.M., Brown, E.M., Fedak, S.A., Woodward, hurbach, C.J., G.D. (1976) J.Cycl.Nucl.Res. 2, 47-56. Spiegel, A.M., and Aurbach, G.D. (1974) J.Biol.Chem. 3, 7630-7636.

1159

The role of GTP in the activation of adenylate cyclase.

Vol. 74, No. 3, 1977 BIOCHEMICAL AND BIOPHYSICAL THE ROLE OF GTP IN THE ACTIVATION RESEARCH COMMUNICATIONS OF ADENYLATE CYCLASE by Alexander Dep...
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