The activation of phosphoglucomutase

Vol.3,No.3

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Sept. 1960

THEACTIVATIONOF PROSPIKGLUCOMUTASg. Cesar Wilsteinl Departzezt of Biochemistry,

Tennis Court Road, Cambridge, Ezgland.

Received August 29, 1960 Phosphogluoomutaee(PGM) is the enayze which catalyses the interconversion of glucose-l-phosphate (G-1-P) and glucoee&phoephate a coenzyme, glucose-1-6-diphosphate, G-l-P. zwcle

(G-6-P).

which is present in the commercial

A pure preparation of the enzyme can be obtained (Najjar,,1948,

Jagannathaz

Luck, 1949a).

and

Mg++ (Jagannathan and Luck, 19%a, Najjar, 1948, Sutherland, 1949).

It require6

rabbit skeletal

from

The pure enzyze requires

1948) and a chelating agent (Najjar,

In the crude preparations the activation

by a

%econd metal" (Cr+++ or Al+++ 1, in addition to Mg++, has also been claimed (Stickland,

1949).

PGt4is isolated ae a phosphoprotein, whose phosphate

exchanges with the'subetrate containingthe characterized

(Jagannathan and Luck, 1949b).

A

pentapeptide

sequence SerP.Ria. in its active centre hae been recently (Milstein

and Sanger,

1960).

PGMhas been obtained by the method of Najjar with a specific of 27 units per mg. of protein component in the ultracentrifuge

(cf.

Najjar,

of acid-stable

It ruas as a single

1948).

under various conditions.

mente have been zade by the procedure of Najjar phosphate formed after

activity

(1948).

Actiritr

deterzining

measure-

the azouut

incubation of the enzyze in the presence

of G-l-P, Mg++ and cysteine or another chelating agent. 1

On leave of absence from the Inetituto Airey)~, Argentina. 292

Naciozal de Hicrobiologia,

Buenos

Vol. 3, No. 3

BIOCHEMICAL

Activation

by

AND

BIOPHYSICAL

RESEARCH

Sept. 1960

COMMUNICATIONS

fwvhehun

The activation

of pure FGM by Hg'+

detected

activity

ie

and Luck

(19491,

is

essenticll;

work

iB,

i%O SigflifiCU%t

This haa been shorn by Jagannathan

in the absence of Mg++.

and in the present

that

with

also

the pure WM prepared

to the method of Najjar. The apparent Hicbaelie constant for -4 magnesiumis 0.5 x 10 . A magnesium-enzymecomplex wa(r also detected by according

equilibrium Activation

dialysis

and the dirrsociation

estimated

ae 0.5

x

Id3

M.

by chelating agents

Chelating agents have two distinct firat

constant

activating

effects

on FGM.

The

is obtained by including a chelating agent in the aesay mixture,

the second by pro-incubating

the enzyme vith EDTAbefore assay (in a very

recent communication in this journal, that preincubation

and

Robineon

and Najjar,

of the enzyme with histidine

1960,

report

and Mg'+ can aleo

activate

FGM).

FGHhas a very high affinity

for heavy metals.

tions cannot be freed from metal6 by simple dialysis water.

In fact "metal free" POM(Milstein,

purification,

can pick

in glass distilled inhibitors

1960) obtained

water during dialysis.

even

by

competes

exchange

‘lmetal

buff ei*

the rabbit

1959),

the presence of Zn*.

with Hg++, with a dissociation

The inhibition technique

(Raaf laub,

constant of 3.9 x 10-14

The lower of the inhibition

con&a&a,

for Cu++ is 2.3 x 10-17. It is

suggerrted

that the high stability

of this type of inactive

complex is responsible for the essential requirement of chelating agent in the

H

also competes with Hg+' but more than one atom of Cu++ per molecule of

enzyme is involved in the inactivation. 5'

by ion

Heavy metale are very strong

by Zn++ and Cu++ has been studied by using the

cu++

again& glass distilled

of FGM. Unlike the yeast FGM (McCoy and Najjar,

zn++

prepara-

up the minute amounts of metal contaminations present

enzyme is completely inactivated

1956).

The purified

assay

mixture.

Partial

activity

ten be obtained in the absence of any

chelating agent by a careful purification In a syetem purified

of all the reagents from metals.

from metals the efficiency

293

of the activation

by

.

Vol.3, No.3

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Sept.1960

cysteine is about tenfold that obtained in a normal aseay, as is shown in Fig. 1.

The reagents were purified

and then washing out all

by shaking with 0.01% dithizone

traces of dithizone

in Ccl4

by shaking the aqueous layer

.five times with Zn++-free CCl4. The enzyme was a %etal free" preparation (Hilstein,

1960).

Fig. 1. mixture.

The activation

of PGHin etandard and heavy-metal-freed

assay

5.1 ug of FGM incubated with 6.5 mMG-l-P and 1.5 mMMgSO4for 5 min. at 30 (final volume 0.4 ml). Curve A. G-l-P and MgS04purified b;r dithizone treatment. Cysteine uas added as indicated, together with metal free tris-HCl to maintain ionic strength and ph. Curve B. As curve A but reagents were not freed from heavy metals.

The suggestion that the activation

by chelating agents is due to

binding of the chelating agent to the active

site of the enzyme has been

advanced for someother enzymes (Haunter et al., 1959).

In FGM, however, this ie not the cane,

metals is a better

1953, Sauer and Rapoport, and removal of the inhibitory

explanation of the mechanismof the eseential activation

by chelating agents. 294

Vol. 3, No. 3

BIOCHEMICAL

Preactivation

by

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EDNA.

Preincubation of PGHwith factor

of 2.5 to 3.

complete after

Sept. 1960

EXYTA

This further

45 min at 30°C.

agents such as cysteine, p

increasee its

activation

specific

activity

by a

is time-dependent, and is

It ia not obtained with other chelating

histidine

or &hydroxyqui.noli.ne

(Fig. 2). 0

/-

E.D.TA.

t-r""

”d’--c ‘3HQ Cyitcine

L5L a- IO Z

Fig. 2.

The activation

of PGMby preincubation with EDNA.

89.2 ug/ml of FGHvere incubated at 30" in the presence of 1 n&l concentrations of the stated substances. In all case.5 pH va8 adjusted to 7.5 vith tris. After the stated times 0.02 ml vere assayed for+$ctivity in the prgsence of 25 mMcysteine, 6.5 mMG-l-P and 1.5 mklMg (2.5 min incubation at 30 1. Point A was determined with enzyme v% ch vas not preincubated vith ElYl!A, in an assay mixture containing 5 x 10 M EDNAin addition to the other components.

Robineon and Najjar (1960) reported that when Mg* is present together with histidine

a similar time-dependent activation

is obtained.

In their

experiment shorter t3mee and a much lover temperature were necessary for complete activation. EDNA

preactivation

can also be obtained with "metal free" RN.

preparationa give an increase in activity activation dialyais

of up to 6 thee.

MTA

Aged

pre-

can be reversed in a time-dependent fashion by restricted against

water and dilution

at O'C.

The process of reversal take6

lees than 10 min at O°C for completiolqby which t%methe specific haa returned to ite original

value.

EDNApreactivation

affects

activity neither

the Michaelis constant for magnesiumnor the sedimentation pattern of the enzyme. 295

Vol.3, No.3

A full

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

account of this work will

I am indebted

be published

to Dr. H. Dixon (F.B.S.)

and totheBritirrhCtnanoil

for

el&en.

him adtioo

foraSoholamhip.

Vol. III pa@ 30l. 0. & Bapaport, 8. (1959); Biochem. 2.. a, 534. SticUand, L.H. (1949); Biochem. J., 44, 191. Sutherland, E.V. (1949); J. Biol. Qmm., l8& l279. Sauer,

296

Sept. 1960

and enootusgemnt