The β-glucoside system of Escherichia coli II. Kinetic evidence for a phosphoryl-enzyme II intermediate

Vol. 45, No. 2, 1971

BIOCHEMICAL

ME

II,

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

f3-GLUCOSIDE SYSTEM OF ESCHERICHIA

KINETIC

CO,51

EVIDENCE FOR A F’HOSPHORYL-ENZYME II Steven

P.

4

Rose

and

C.

Fred

INTERMEDIATE*

Fox?

Department of Biochemistry, University of Chicago, Chicago, and Department of Bacteriology and the Molecular Biology University of California, Los Angeles, California

Received

August

%V$ARY, enolpyruvate properties

The

9, 1971

B-glucoside dependent indicative

In Escherichia port

and

the

specific phosphotransferase of a “Ping-Pang” coZi

phosphorylation

The

phosphorylation

PsHPr

concentration shows

that

expected

enzyme

the of

varied

kinetic

a two

at

substrate

I lbgz in

constant

properties

’

of

reaction

in PQHPr )

enzyme

Kundig,

the

Ghosh

following

is

trans-

II

(I)

and

of

Roseman

scheme.

+ pyruvate

S-glucoside-6-P

the

kinetic

values

HPr

or

versa.

the

enzyme

vice

sequence

the phosphocoZi exhibits

catabolism

by

described I

of

specific

described

enzyme

,

S-glucoside

a B-glucoside

is

inconsistencies was

in

initially

+ B-glucoside

apparent

I I (enzyme I lbgZ) system of Eacherichia kinetic mechanism.

step by

process + HPr

enzyme

first

system

P-enolpyruvate

We noted

the mediated

phosphotransferase

(2) *

Illinois, Institute,

II

+ HPr obtained

where

A systematic

mediated

involving

substrate study

reaction

are

a substituted

those

enzyme

intermediate.

MmrODS,

k The f

In

membranes

initial

Address reprint of California,

7 Current Los

paper

derived

in

this

from

series

requests to Los Angeles,

strain

is

reference

C. F. Fox, California

address : Department of Angeles, California 90024.

W1895Dl-bgZ+c,

376

single

activity

I.

Department 90024.

Biological

the

Chemistry,

of

Bacteriology,

University

University

of

California,

Vol. 45, No. 2, 1971

catalyzing this

TEG’ strain

scribed

phosphorylation

and (1).

BIOCHEMICAL

for

The

the

(3-4).

I lbg2

contained

bovine

serum

of

enzyme

and

HPr

are

present

form to

and

of an

except

ml

of

the

twice

washed

were system

TEG

entire

was

(5)

containing

Abbreviations used I-thio-b-D-glucopyranoside; D-glucopyranoside;

by

incubated

at by

been

of

de-

purified),

described

in by

MM MgClz,

the

source

2.5

of

detail

enzyme

mg/ml

of

all the

of

was

v/v

20

is

washed

with

Triton

6.5 X-100

in

the

membranes

minutes.

The

procedure

mixed of

II bg2 ,

HPr

of

C for

pg

P-enolpyruvate

the

addition

28”

174

enzyme

1 and

a published

5 ml 6:7

(I), ml

of

(Rohm

and

Haas)

counting.

I/

1

be

growth

5 mM 2-mercaptoethanol,

essentially

of

has

(40-fold

Enzyme

initiated

I 8

FIGURE 1, Double reciprocal HPr is varied at constant phosphoryl I/v, nmoles-l 250 IJM (0) and 50 IIM (0).

50

as

determined eluate

SC intillation

that

and

Dowex-I

solution

pH 7.6,

for

phosphorylation

concentrations.

reactions

phosphoryl

TEG

membranes

so

I will

10 mM KF,

excess

complete

enzyme

for

of

procedure membranes

purified) system

indicated

sufficient The

for

assay

The

washed for

(150-fold

50 mM Tris*HCl

at

the

twice

mM P-enolpyruvate,

TEG

otherwise

toluene

0.4

of

Patterson-Green and

The

pg

P%HPr.

that

HPr

II

in

formation

and

albumin, I,

of procedures

A,

12.5

II bg2 .

enzyme

preparation

purification

phosphoglucosidase elsewhere

is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

16

24

32

~

[HPrl

W

mediated plots for the enzyme II TEG concentrations. Units are I/[HPr], TEG formed in 20 minutes. Concentrations

are: PNPG-6-P,

TEG,

ethyl-1-thio-D-D-glucopyranoside; TPG-6-P, TPG-6-phosphate; PNPG-6-phosphate.

377

PNPG,

reaction where mg-l ml and of TEG are

TPG, phenylp-nitrophenyl-8-

Vol. 45, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I/ [TEG] FIGURE 2, Double reciprocal plots for the enzyme I lbgZ mediated TEG is-yaried at constant HPr concentrations. Units are l/[TEG], nmoles of phosphoryl TEG formed in 20 minutes. Concentrations 32.5 w/ml (01, 65 US/ml (0) and 325 pg/ml (A).

RESULTS, the

Double

enzyme

reciprocal

I lbgZ

Figures

1 and

strates

is

reaction

2.

The

varied

indicative

of

steady

state

lation

(6),

plots

at

mediated parallel

where

v,

varied

displacement

equation

=

a,

*

K a’

a,

limiting

from in

the Figures

calculated and

0.17

or

are

obtained

when

lines

of

the

vs.

one

for in

of

the

substrate

kinetic

mechanism,

I/[TEG]

presented

other

“Ping-Pang”

kinetic

l/v

membranes

be

primary 1 and for

1 VI V1

sub-

are

mechanism. using

The

Cleland’s

formu-

> represent of

Michaelis

plotting

each

Kb can

-

and

Kb’

By for

and

this

concentration

repsectively.

VI

washed

or

for

+

b,

of

obtained

l/[HPr]

is

substrate,

constant

by

vs.

concentrations

K l/v

I/v

straight

constant

a double rate

of

reaction where mM-’ and l/v, of HPr are

I/a

vs. of

calculated.

2, KHpr

mM respectively

Using

of

l/a

values at

vs. of

unvaried

substrate,

an

b.

From

and

at

b,

and

of

Frcm

the

0.46

mg per

K,

separate ml

can

be

I/b

vs.

),

and

saturating

P%HPr

(limiting

l/v’,

calculated depicted

respectively

KpsHpr

is

(v’)

experiments

(lrmltlng

378

velocity

velocity of

the

Michaelis

maximal

plots VI,

of

limiting

maximal

secondary

. . .

concentration

the

apparent

value

l/v.

TEG KTEG

of

l/v,

this

0.35

saturating for

velocity,

constant

concentration

plot

the

initial

were values

of

KTEG).

0.14

Vol. 45, No. 2, 1971

TABLE

1,

BIOCHEMICAL

Irreversibility

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

R-glucoside

phosphorylation

Incubation (minutes)

System

mediated

time

Change

in at

Complete

120

< 0,005

2.

Add

120

< 0.005

?d.

Add

HPr, enzyme and P-enolpyruvate

II bgl

enzyme

absorbance 420 nm

1.

HPr

by

I

2.86

5

The complete system (0.5 ml) contained 1.4 mg of enzyme II bgz, 193 vg of phospho-f3-glucosidase A, 1 mM PNPG, 1 mM TPG-6-P (3), 50 PM MgC12, 10 mM KF, 75 r&I Tris*HCl of pH 7.6, and 7.5 mM 2-mercaptoethanol. Enzyme II mediated transfer of phosphate from TPG-6-P to PNPG would be detected by the increase in absorbance at 420 nm resulting from the phospho-f3-glucosidase catalyzed release of p-nitrophenol from PNPG-6-P. Where indicated, the reaction vessels also contained 7.5 mM P-enolpyruvate, 64 pg of HPr, and 44 pg of enzyme I. Reactions were incubated at 28” C and terminated by the addition of 0.05 ml of 50% (W/V) trichloroacetic acid. After a 10 minute centrifugation at 10,000 x g, 0.3 ml of the suoernatant solution was added to 0.6 ml of 2 M KOH. and the absorbance compared with that of a similarly treated sample identical-to the complete system above, but for the omission of TPG-6-P.

The

inabi

1 i ty

between

TPG-6-P

and

reaction

is

reaction

exceeding

DISCUSSION, here

is given

of

phosphate

irreversible

in

enzyme

PNPG

A sequence

Figures from in

to

I)

of

the

of

steps

3.

The

Figure 1 and

2.

phosphoryl

catalyze

shows

irreversible that

in

I lbgZ

(Table

essentially

given

is

of

in back

that

the

reaction

enzyme

II

at

with

evidence data

the by

for in

Table

to

the

group I I bgl

enzyme

extracts,

consistent

The

a phosphoryl

rate

of

least the

I indicate glycoside

the

forward of

magni

findings

substrate

tude.

reported

kinetic that

e

mediated

4 orders

kinetic

a “Ping-Pang”

exchangl

mechanism the

transfer is

essentially

extracts.

PwHPr-T;

E,

(P-HPr*E& HPr -k ro osed scheme for ~~?!~x?‘fo~ ditai 1s Symbols R-Glu, B-glucoside; i-Glu-6-P,

-7;;

/3-Glu-6-P

(PwE@-GId P-E,

-k?

P-Glu

B-glucoside phosphorylation used are PcHPr, phosphoryl-HPr; B-glucoside-6-P.

379

by

enzyme Eli,

II bgl enzyme

. II;

Vol.

45,

Hays

No.

2, 1971

Our

kinetic

and

BIOCHEMICAL

results

Roseman

transport

which

(and

obtained

the

S.

induced

sugar

a second

binding,

phosphorylated

in not

by

the

no

soluble

be

the

principal

coupled

to

depicted

to

of have

more

than

their

in

3 of our

mediated

by

data

membrane

the

that

the

3 is

indicate

stems

8-glucoside

that

a phosphoryl-enzyme

1.

2.

by

Proc. Nat. S. Roseman,

an

be

the

detected

however,

do

system

their

apparent

I I bgl

enzyme site

for linked

(8) II

for

binding

role and

is

intimately

phosphorylatior R-glucosides

transport

is

likely

I I intermediate,

supported by USPHS grant AM-10987. trainee supported by grant GM-00090, and Research Career Development Award GM-42358. discussion.

Fox, C.F. and G. Wilson, Kundig, W., S. Ghosh and

from

binding

ACKNCNL~~NTS, predoctoral of USPHS for helpful

data,

can

Kaback

responsible enzyme

extracts, on

zuc

III

is

protein.

mediated

also

factor

phosphorylation.

transport If

Figure

for

II

which

auTeu.s

phosphorylating

bound

enzymes

between

zac)

III

In S.

kinetic

B-glucoside

substrates

proposed

step

the

one in

phosphorylation.

transport, be

that

Our

Data

phosphorylation,

to

extracts.

(7).

determines

(factor

G-glucoside

linked

formation

presumably

protein

corresponding

sonic

interest

transport (9)

of

P-enolpyruvate

complex

which

Simoni,

auTeu.s

substrate.

For

protein

possibility of

glycoside

Nakazawa,

for

ternary

induced

the

COMMUNICATIONS

StaphZocacctls

II zac)

protein.

inducible

composed

Roseman

for

a soluble

the

The

and

of

scheme

in

indicate

specifically

PsHPr,

those

complex

(enzyme

RESEARCH

with

lactose

system

protein

fraction

preclude

could

in

is

I I I zac hand,

a more of

aureus

BIOPHYSICAL

contrasted

indicate

a specifically

other

be

phosphorylation)

with

factor

may

AND

S.P.R. was a USPHS C.F.F. the recipient We thank Dr. F. KBzdy

Acad. Sci. U.S. 59, 988 (1968). Proc. Nat. Acad. Sci. U.S. 52, 1067

(1964). 3. 4.

2: 7.

8. 9.

Wilson, G. and C.F. Fox, manuscript in preparation. Ph.D. Dissertation, The University of Chicago (1970). Rose, S.P. and C.F. Fox, manuscript in preparation. Ph.D. Dissertation, The University of Chicago (1971). Patterson, M.S. and R.C. Green, AnuZ. Chem. 37, 854 Cleland, W.W., Biochim. Biophzjs. Acta 6-7, 104 (1963). Nakazawa, T. , R. D. Simon i , J . B. Hays and S. Roseman,

Commun.42, 836 (1971). Kaback, H.R., J. BioZ. CTzem.243, 3711 Roseman, S., J. Gen. Physioi!. 54, 138s

380

(1968). (1969).

See

also,

Wilson,

See

also,

Rose,

G., S.P.,

(1965). Biochem.

Biophys.

Res.