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
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
