Galactosyl transferase: The role of manganese ions in the mechanism

Vol.

68, No.

4, 1976

BIOCHEMICAL

GALACTOSYL

AND

TRANSFERASE: IN A.D.

Received

THE THE

December

ROLE

RESEARCH

OF

COMMUNICATIONS

MANGANESE

IONS

MECHANISM

Tsopanakis

and

Department of Leeds, 9 Hyde

University

BIOPHYSICAL

D. G.

Herries

of Biochemistry Terrace, Leeds

LS2

9LS,

U.K.

2.1975

Double reciprocal plots for galactosyl tronsferase with UDP-golactose varying at several fixed Mn2’c oncentrotions are a series of straight lines. Different laboratories disagree as to whether the intercepts on the l/v axis are independent of Mn2+ or not. PbCl2 added in a fixed proportion is shown theoretically to be incapable of introducing such a dependence A mechanism derived from extensive kinetic studies is on total metal ion concentration. presented, with alternative pathways for free UDP-galactose and the Mn2+complex as substrates, following obligatory Mn 2+ addition, and the conflicting results from different laboratories are explained on the basis of o different flux through the alternative pothways under different conditions. INTRODUCTION Galactosy

I transferase

UDP-galactose In the

lactating

transfer

carried HCI

by

buffer.

and

(2) by

catalyses

the

Morrison

and

the

and studies

on specific

Powell

and

Brew

the

enzyme

presence

inhibition (1) at 30°C

under

different were

reaction:

as lactose

studies and

molecular

weight

forms

(3) using

a different

assay

enzyme

bovine

system

galactosyl

2.4.1.22). bovine

milk

were

M N-ethylmorpholine-

pH 7.4,

milk

of the

from

in 0.25

(37’C, human

(EC

enzyme

pH 8.0

on the

enables

synthase

on the

conditions

made

+ UDP.

of a-lactalbumin

is known

Ebner

buffer)

following

N-acetyllactosamine

where

dead-end

sulphonate

Brew

enzymes

gland,

to occur,

Similar

linol-propane and

mammary

velocity

out

2.4.1.38)

+ N-acetylglucosamine+

to glucose Initial

(EC

milk

from

that

0.05

M 3-CN-morpho-

by Khatra, and

Herries

colostrum

of Morrison

and

Ebner. With Ebner against

N-acetylglucosamine

found

that

the

!JJDP-galactosel

as acceptor

intercept -1

on the was

vertical

independent

Copyright 0 I976 by Academic Press, Inc. All rights of reproduction in any form reserved.

in the axis of total 1102

absence in double Mn2+

of a-loctalbumin,

Morrison

reciprocal

plots

concentration

and

of velocity interpreted

and -1

Vol.

68, No. 4, 1976

this

to mean

that

BIOCHEMICAL

the

galactosyl

equilibrium

conditions,

cycle

1, scheme

(Fig.

dependence

of the

the

equilibrium

not

as the

scheme

species,

plots

on total

as a Mn

2+ and

2+

2+

Mn

and

not

Khatra

Mn

of Morrison but

Geren

and

ence

in the

results

found

their

MnC12

which

had

ions,

they

enabling

were

able

them

of the

sulphydryl

in a constant intercept

to put

was

complex,

last

put

forward

a complex, turn

of the

and Brew have

Powell which

and

COMMUNICATIONS

form

at each

et al and

Ebner,

ions

dissociate

-concentration,

-UDP

(4) have

by the

group

(5). by theoretical

proportion

cannot

on extensive

groups. been

the

MnC12

ion

recently

By adding

treated

with

a dependence

to show

metal

two

previously

forward

group’s

on total based

Ebner

to create

other

We wish

ation

In such

need

RESEARCH

in the

a

with

by releose

catalytic

catalytic found

is incompatible

explained

under

2+

of Mn

cycle

(Fig.

,

1,

2). Geren,

tion

Mn

BIOPHYSICAL

protein

2+

which

intercept

mechanism

free

transferose

from 3).

AND

view by

lead,

which

concentration studies

MATERIALS

of the AND

for

mol)

could

inhibit

that

to propose reaction

the

the

of the

Mn was

double

via

of such

at higher

to Pb

to contaminaits active

2+

site

an inhibitor plot

mechanistic Mn

2+

concentration,

reciprocal

an alternative rate

2+

due

enzyme

presence

of PbC12 to remove

on total in results

a dependence and

(mol

intercept

difference

considerations introduce

kinetic

the

0.001%

of the differ-

diphenylthiocarbazone

of the

that

an explanation

explan-

concentrations.

METHODS

3-(N-morpholina)-propanesulphonic acid (MOPS) was obtained from BDH Chemicals Ltd recrystallised before use. MnCl2 was BDH An a I a R grade (heavy metal content 0.0005%, UDP-galactose and N-acetylglucosamine were from Sigma, UDP-[‘4Clgalactose w/w). from Radiochemical Centre, Amersham, or New England Nuclear Corporation, and Sepharase 4 B from Pharmacia. Galactosyl transferase was prepared from bovine milk or colostrum by affinity chromatography as described by Barker et al (6) using Sepharose 4 B covalently linked to UD P-hexanolamine. The purity of the enzyme>as checked by gel electrophoresis in the presence of dodecyl sulphate as outlined by Powell and Brew (3). Velocities were measured as described by Tsopanakis and Herries (7) except that the reduction in the concentration of Mn2’ due to complex formation with the buffer was allowed for. Stability constants were determined by electron spin resonance spectroscopy and free concentrations of the and

1103

Vol.

68, No.

4, 1976

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

components of the enzyme system evaluated by an iterative were expressed as nkat per litre. Kinetic data were analysed by fitting appropriate rate written by one of the authors in Algol 60 for the ICL 1906A Leeds. The principal program used was constructed around Library program EO4GAA which uses the function minimisation RESULTS Scheme

3 of Morrison

is identical enzyme

except again

formed

that

UDP

1).

The

to double

reciprocal

giving

Eqn (l),

constant, the

(Fig.

and

term

ponsible intercept If the

K,,(M for

in the

Ebner

initial

shown

intercept

lack

of dependence

of the

plot

of

velocity the

in Fig.

2.

upon

against

mechanism

iu E

EM

reference

as the

with

EMA (l),

in Fig.

for

is absent,

B

P

R

C

B

P

5

and and

-1

so that

scheme 3

A

-UDP,

*

producing

mechanisms

Ebner’s

it is this

of the

EM

EMA

EMAB

reference

(Z),

scheme 2

be trans-

mechanism, which

is res-

is shown

by the

five

enzyme

ER

E

R

M

species

* C

B

P

scheme A2 A M

* scheme A3

M

C

B

P

R

scheme M

Various possible mechanisms for the galactosyl B, C, P, Q, R, S represent enzyme, Mn2+, Mn2+-UDP-galactose, N-acetyllactasamine, respectively.

1104

free

kept

equilibrium

which

(2)

.

each

scheme Al

Figure 1. E, M, A, cosamine, (Mn2+)2-UDP

programs of Group (8).

et al -the

may

absence

ion concentration

E E

2 of Khatra

of B (N-acetylglucosamine)

In Morrison

EM

2+

these

concentration

metal

Scheme

Mn

equations

is modified

EMQ

Velocities

equations with computer computer at the University the Nottingham Algorithms method of Marquardt

1.

complex,

[UDP-galactose]

EMAB

program.

DISCUSSION

is shown

expression

the

equilibrium

(1)

is released

form,

l/v0

AND

computer

COMMUNICATIONS

transferase reaction. UDP-golactose, N-acetylgluUDP, Mn2+-UDP, and

(E,

Vol.

68, No. 4, ‘976

Scheme

BIOCHEMICAL

3, Morrison

‘/v

8, Ebner

= (l/v)

0

In scheme

(1) and

(KiaKdB 3 the

AND

BIOPHYSICAL

scheme

2,

+ Ka) (1 + K;,,/M)

intercept

term

K,/M

RESEARCH

Khatra

--et al

(‘/A)

(2)

+ (l/V)

COMMUNICATIONS

:’

(KdB

+ 1 + KI+/M)

(1)

is absent

Figure 2. Initial velocity equations for the first two mechanisms of Fig. 1 . M, A and B are described in Fig. 1. V is the maximum velocity; Kim and K. are dissociation constants for the reaction of M with E, and A with EM, ” respectively; Km, K, and Kb are Michaelis constants for M, A and B, respectively.

EM,

EMA,

ciation

EMAB

and

constants

EMQ)

can

respectively

K,

of the concentration

of M,

‘/v 0

(KiaKdB

= (‘/V)

form

say

a dead-end

to Kg,

and

then

Eqn (2)

a,

+Ka)

complex

if I is always can

(1 +Kin(M

+ (l/V)

(Kb/B

with

+l

an inhibitor

present

I, with

at a constant

proportion

be written.

+ SiJK,

+ alvl/B

+ ab#K2)

Kb/K3

+

(l/A)

1)

kdK4 + kJ”5

(2)

k

7 +k9 (k7

and

ively

k

are

9

rate

to release The

expression

Thus

higher

as an inhibitor. will

reciprocal

to the ri& Such to combine Geren

The

individual

of the

with

l/v0

function

mechanism,

of M,

i.e.

a lower

hand

constant

corresponding

as M increases,

maximum

velocity,

is a hyperbolic

as M varies.

as M increases,

respect-

The value

function

effect

so that of M,

will

be to cause

an inhibitor,

the

M is acting

such

on a set of lines

of M,

as if M were

so will

its

that

in the double them

to move

or to cross

each

other

axis.

is not enzyme

et -- al (4) show

other

at a different graph

behaviour

on the

in the

UDP).

of M produce

a minimum

each up the

values

steps and

is a linear

slope

pass through plot,

progressively

for

of N-acetyllactosamine

intercept

intercept.

value

constants

disso-

shown

forms both

by

Pb2+

’tons,

in the

way

described

intercept

and

slope

although for

decreasing

tration.

1105

it is reasonable the

inhibitor,

with

increasing

to expect I.

The metal

Pb

results ion

2+

of concen-

Vol. 68, No. 4, 1976

If purely combine

with

competitive

inhibition

free

and

to KS removed on the too, of the

by

and

Morrison

We same

therefore and and

have

were

that

(3)

is not

(1).

out

as Khatra

stability determined

MnCI

constant

2

studies have

concentrations

spin used

and

Mn

in any

resonance were

2+

0

= .

that

set of velocity

In scheme

A2,

Klrn

the

difference

2+

into

Mn

between

at pH 7.4

of 2.0

the

free

LKm

2+

account

intersect

This

behaviour,

include

the

by Khatra

dependence

et al --

it cannot

them

be due

The Mn

concentrations the

M

to

2+

-1

ion

(K’

m

and

Mn

total

L)M

remained Mn

2+

.

Rate

+ KaM2/K C

+

Km = 0;

in scheme

+ V3KaM)

A4,

Km = Klrn

= 0

Figure 3. Initial velocity equations for schemes Al to A4 of Fi$+l, expressed in terms of total UDP-galactose concentration (Ao) and free Mn (M). “1 is constants for A and M, respectively, the maximum velocity and Kar Km are Michaelis if the lower pathways of the schemes are missing; V3 is the maximum velocity and Kc, K’, are Michaelis constants for C and M, respectively, if the upper constant of M from pathways of the schemes are missing; Kim is the dissociation EM, and L is the dissociation constant of M from C. Except for Kim and L, the constants are functions of B.

1106

.

of UDP-galactose

“lKc A3,

2+

respectively

concentration

+

the

of complexes

and 58 M-l

at constant

+

under

formation

ranges

V,M(L

in scheme

(2)

between

UDP-galactose

C

= 0;

K2

will

in results

at pH 8.0,

at higher

aM) “lK

used

measurements

(Wo) “,M(L+“f

not

M&l2

and

Ka (L + M)(Kim + M) l/v

is absent.

only

I will

containing plot

of the

spectroscopy.

such

reciprocal

found.

of Pb

taken

(pH-dependent)

by electron

3%

and

terms

authors

for

inhibitor

enzyme.

kinetic (l),

the

as it does

these

COMMUNICATIONS

the

inhibitor

et -- al (4),

effect

the

sulphonate

constants

to within

the

double

the

contamination

with

--et al

as when

the explanation

extensive

Eqn (2) with

in the

which

Whatever

complexes

lines

of Geren

conclude

morpholinopropane

Apparent

results

RESEARCH

to M is considered,

exist.

way

concentration,

Ebner

carried

will

ion

Brew

of dead-end

conditions

between

and

on metal

Powell

formation

the

K,

same

BIOPHYSICAL

respect

a set of

in the

with

AND

with

only

that

exactly

consistent

must

and

predicts

axis,

intercept

We

enzyme

then

l/v0 is not

BIOCHEMICAL

(3)

Vol.

68, No. 4, 1976

equations

BIOCHEMICAL

derived

in terms

UDP-galactose

(Ao)

assumed

constant

where

Because

the

complex

of this

work

will

could

not

experimental predict of straight

a plot

as M increases. ter-assisted

shown

of l/v0

without Fig.

matching

with

the

arranged

in Fig.

the

2+

Mn

initial

likely

a common 4 illustrates

Al

scheme

Al

A4 were

intersection

experimental

fixed

point.

where

the

points

C is the

scheme with not

are

at several

this,

constant

we were

to free

that

equations

l/A0

COMMUNICATIONS

be transformed

that

to &I).

found

A3 and

velocity

therefore

concentrations,

substrate

We

Schemes

could expectation

1 (schemes

less

RESEARCH

to total

M could

be

experimentally.

at higher

against

of the

species

elsewhere.

out. The

BIOPHYSICAL

as an alternative

although

be ruled

lines

activity

be reported

results. that

M was

functions

results

individual

Mn 2+ (W,

total

schemes

which

experimental UDP

free

of enhanced

particularly tose

and

of the

AND

lines

to the

Mn

2+

-UDP-galac-

UDP-golactose.

The

A2 wos

with

release

consistent

of a product,

consistent

with

shown

in Fig.

values

of M will

3.

Slopes

and

intercepts

drawn

are

calculated

form

led to consider

of Eqn

the

(Mn2+),-

certain All

results

of the

schemes

consist will

(3) appropriate

of a series decrease

by the

compu-

to scheme

A2.

I

I -5

I 5

I 0 l/UDP-galoctose

I 10

I 15

mtd

Figure 4. The effect of UDP-galactose concentration on the rate of galactosyl transfer to N-acetylglucosamine (constant at 10 mM) at different fixed concentrations of free Mn2+ ion, There is no common intersection point for the lines. Concentrations of free Mn2+ (mM) were d 0.362, m 0.453, v 0.604, A 0.906, l 1.81, 0 2.27, 3.01, 4.54, 9.09. The enzyme was prepared from bovine milk.

V

A

1107

0

Vol.

68, No.

Since woy

4, 1976

we

which

not

is Morrison to scheme

intercept

section

A4.

predominates

pH 7.4,

a significant

(2) were

centration.

The

3, we

for

the

dependence

they

plot.

appear

Although

Kc

lorge;

see

of using

with

through

the

total

Mn

at the lower

2+

of temperature

discuss

Morrison

of the upon lines and

3), Mn

takes

high place

the

Ebner’s

of inter-

repeated

by

pathwoy

in scheme

to a lower

Mn 2+-UD

less than buffer

chemical

results

o common

results,

and

poth-

pathway,

have

due

upper

concentration

upper

up to 10 mM, and

2+

concentration

relatively

concentrations

and

Ebner’s the

the

alternative Mn

probably

2+

A4,

do not

at pH 8.0

pathway

difference

scheme

the

Fig.

0 total

by complexation

flow

that

COMMUNICATIONS

ogoinst

Morrison

to do so.

by assuming

RESEARCH

moy

is responsible

further

effects

Scheme

introduction

as a result

obtained

evidence

the

V3 small,

reduced

the

that

(i.e.

effectively

BIOPHYSICAL

It is evident

be explained

concentration

--et al

Ebner’s

can

golactose

with

reciprocal

in practice

--et al (4),

AND

here

and

in the double

point,

Geren

not

concerned

uses C as substrate,

the

A4

ore

of which

in relation

BIOCHEMICAL

P-

1 mM,

concentration.

At

the

results

of Khotra

and

a lower

buffer

nature

of the

buffer

concan-

be predicted. Acknowledgements

We are grateful trometer.

to Dr.

P. F.

Knowles

for

allowing

us to use his electron

spin

resonance

spec-

REFERENCES 1. 2. 3. 4. 5. 6. 7, 8.

Morrison, J.F., and Ebner, K.E. (1971) J. Biol. Chem. 246, 3977-3984. Khatra, B.S., Herries, D. G., and Brew, K. (1974) Eur. J. Biochem. 44, 537-560. Powell, J.T., and Brew, K. (1974) Eur. J. Biochem. 48, 217-228. Geren, C.R., Geren, L.M., and Ebner, K. E. (1975) Biochem. Biophys. Res. Commun. 66, 139-143. and Ebner, K.E. (1974) J. Biol. Chem. 249, 6992-6998. Magee, S.C., Barker, R., Olsen, K.W., Shaper, J.H., and Hill, R.L. (1972) J. Biol. Chem. 247, 7135-7147. Tsopanakis, A.D., and Herries, D. G. (1975) Eur. J. Biochem. 53, 193-196. Marquardt, D. W. (1963) J. Sot. Ind. Appl. Math. 11, 431-441.

1108