Metal ion binding to D-xylose isomerase from Streptomyces violacoeruber

Abstract

The binding of two activating cations, Co2+ and Mg2+, and of one inhibitory cation, Ca2+, to D-xylose isomerase from Streptomyces violaceoruber was investigated. Equilibrium-dialysis and spectrometric studies revealed that the enzyme binds 2 mol of Co2+/mol of monomer. Difference absorption spectrometry in the u.v. and visible regions indicated that the environment of the first Co2+ ion is markedly different from that of the second Co2+ ion. The first Co2+ appears to have a six-co-ordinate. The conformational change induced by binding of Co2+ to the first site is maximum after the addition of 1 equivalent of Co2+ and yields a binding constant greater than or equal to 3.3 x 10(6) M-1. Binding of Co2+ to the second, weaker-binding, site caused a visible difference spectrum. The association constant estimated from Co2+ titrations at 585 nm agrees satisfactorily with the value of 4 x 10(4) M-1 obtained from equilibrium dialysis. Similarly, the enzyme undergoes a conformational change on binding of Mg2+ or Ca2+, the binding constants being estimated as 1 x 10(5) M-1 and 5 x 10(5) M-1 respectively. Competition between the activating Mg2+ and Co2+ and the inhibitory Ca2+ ion for both sites was further evidenced by equilibrium dialysis and by spectral displacement studies.

Full text

Biochem.
J.
(1988)
250,
285-290
(Printed
in
Great
Britain)
Metal
ion
binding
to
D-xylose
isomerase
from
Streptomyces
violaceoruber
Mia
CALLENS,*t
Peter
TOMME,*
Hilda
KERSTERS-HILDERSON,*
Rita
CORNELIS,t
Werner
VANGRYSPERRE*
and
Clement
K.
DE
BRUYNE*
*Laboratorium
voor
Biochemie,
K.
L.
Ledeganckstraat
35,
and
tLaboratorium
voor
Analytische
Scheikunde,
Proeftuinstraat
86,
Rijksuniversiteit
Gent,
B-9000
Gent,
Belgium
The
binding
of
two
activating
cations,
Co2l
and
Mg2",
and
of
one
inhibitory
cation,
Ca2",
to
D-xylose
isomerase
from
Streptomyces
violaceoruber
was
investigated.
Equilibrium-dialysis
and
spectrometric
studies
revealed
that
the
enzyme
binds
2
mol
of
Co2+/mol
of
monomer.
Difference
absorption
spectrometry
in
the
u.v.
and
visible
regions
indicated
that
the
environment
of
the
first
Co2+
ion
is
markedly
different
from
that
of
the
second
Co2"
ion.
The
first
Co2'
appears
to
have
a
six-co-ordinate
octahedral
symmetry,
whereas
the
symmetry
of
the
second
Co2+
is
less
evident,
being
four-
or
five-co-ordinate.
The
conformational
change
induced
by
binding
of
Co2+
to
the
first
site
is
maximum
after
the
addition
of
1
equivalent
of
Co2'
and
yields
a
binding
constant
>
3.3
x
10i
M-1.
Binding
of
Co21
to
the
second,
weaker-binding,
site
caused
a
visible
difference
spectrum.
The
association
constant
estimated
from
Co2"
titrations
at
585
nm
agrees
satisfactorily
with
the
value
of
4
x
104
M-1
obtained
from
equilibrium
dialysis.
Similarly,
the
enzyme
undergoes
a
conformational
change
on
binding
of
Mg2+
or
Ca2",
the
binding
constants
being
estimated
as
1
x
105
M-1
and
5
x
105
M-1
respectively.
Competition
between
the
activating
Mg2'
and
Co2'
and
the
inhibitory
Ca21
ion
for
both
sites
was
further
evidenced
by
equilibrium
dialysis
and
by
spectral
displacement
studies.
INTRODUCTION
D-Xylose
isomerases
(EC
5.3.1.5)
catalyse
the
conver-
sion
of
D-xylose
and
D-glucose
into
D-xylulose
and
D-
fructose
respectively,
and
have
attracted
considerable
interest
for
the
production
of
high-fructose
corn
syrup
and
ethanol.
D-Xylose
isomerases
are
dependent
on
the
bivalent
cations
Mg2",
Co2"
or
Mn2"
for
catalytic
activity.
These
metal
ions
activate
D-xylose
isomerases
to
different
extents,
depending
on
the
origin
of
the
enzyme
and
the
substrate
(Chen,
1980).
In
the
specific
case
of
D-xylose
isomerase
from
Streptomyces
violaceoruber,
the
highest
activity
towards
D-xylose
and
D-glucose
is
obtained
with
Mg2+,
although
Co2+
and
Mn21
support
activity
with
various
degrees
of
efficiency
(Callens
et
al.,
1986).
Furthermore,
stabilization
of
D-xylose
isomerase
by
metal
ions
has
been
recognized
for
many
years
(Chen,
1980).
The
results
with
this
typical
D-xylose
isomerase
are
consistent
with
Co2`
being
superior
to
Mg2'
as
protector
against
thermal
denaturation
(Callens
et
al.,
1986).
Although
many
isomerases
have
been
characterized,
information
about
direct
metal
ion
interactions
is
very
restricted.
As
reported
by
Danno
(1971),
Co2+
is
bound
to
D-xylose
isomerase
from
Bacillus
coagulans
in
a
molar
ratio
of
1
equiv./monomer.
Schray
&
Mildvan
(1972)
published
binding
data
from
n.m.r.
studies.
The
data
fitted
to
an
equation
that
assumed
independent
binding
of
Mn2+
at
approximately
three
tight-binding
sites/
molecule
of
protein
with
K
=
3.7
x
104
M-1
and
approxi-
mately
21
weaker
metal-ion-binding
sites/molecule
of
protein
with
K
=
8.5
x
102
M-1
for
D-xylose
isomerase
from
Streptomyces
sp.
A
similar
study
by
the
same
authors
with
D-xylose
isomerase
from
Lactobacillus
brevis
indicated
2.1
tight
Mn2+-binding
sites
(K
=
1.0
x
105-1.6
x
105
M-1)
and
4.4
weaker
Mn2+-binding
sites
(K
=
5.0
x
104
MW1).
D-Xylose
isomerase
from
Strep-
tomyces
griseofuscus
was
found
to
contain
four
Co2"
ions/molecule
of
enzyme.
One
of
the
four
Co2"
ions
was
very
tightly
bound
to
the
enzyme
and
had
an
essential
role
in
maintaining
the
ordered
conformation,
especially
the
quaternary
structure,
of
the
enzyme
(Kasumi
et
al.,
1981,
1982).
Danno
(1971),
Young
et
al.
(1975)
and
Kasumi
et
al.
(1982)
all
postulated
that
the
role
of
the
bivalent
cations
in
the
active
ternary
metal
ion-substrate
complexes
appears
to
be
structural
rather
than
being
directly
involved
in
the
catalytic
process.
In
the
present
paper
we
report
our
direct
binding
studies
of
Co2",
Mg2+
and
Ca2+
with
the
use
of
equilibrium
dialysis
and
difference
absorbance
spectrometry.
To
deduce
the
structure
of
the
metal
ion
environment,
Co2+-
substituted
isomerase
is
preferred,
since
Co2+
ions
appear
to
be
more
suitable
as
a
spectrometric
probe.
EXPERIMENTAL
Materials
The
cation
salts
CoCl2,6H20,
MgCl2,6H20
and
CaCl2,2H20
were
pro
analysi
products
from
E.
Merck,
Darmstadt,
Germany.
D-Sorbitol
dehydrogenase
from
sheep
liver
and
NADH
(grade
I)
were
obtained
from
Boehringer,
Mannheim,
Germany.
Chelex
100
is
an
analytical-grade
chelating
resin
from
Bio-Rad
Labora-
tories,
Richmond,
CA,
U.S.A.,
and
Nitroso
R
salt
was
obtained
from
Sigma
Chemical
Co.,
St.
Louis,
MO,
U.S.A.
All
other
chemicals
were
of
analytical
grade.
t
To
whom
correspondence
should
be
addressed.
Vol.
250
285

M.
Callens
and
others
Table
1.
Cobalt
measurements
with
Nitroso
R
salt:
incubation
of
enzyme
with
different
Co2+
concentrations
D-Xylose
isomerase
(200
#M
active
sites)
was
incubated
with
various
Co2+
concentrations
in
0.01
M-triethanol-
amine
buffer,
pH
8.0,
at
35
°C
for
4
h.
Free
Co2+
measurement
(#M)
Added
Co2+
Enzyme
Enzyme
Co2'
deficiency
(#M)
absent
present
(equiv./monomer)
60
80
100
120
160
200
250
300
400
61
80
99
119
160
202
248
301
400
8
6
11
14
18
31
64
99
204
0.27
0.37
0.44
0.53
0.71
0.86
0.92
1.01
0.98
Enzyme
preparation
and
assay
D-Xylose
isomerase
from
S.
violaceoruber
(L.M.G.
7183)
was
purified
to
a
homogeneous
state
as
described
previously
(Callens
et
al.,
1985).
The
enzyme
concentrations
were
routinely
estimated
from
the
u.v.
absorption
at
280
nm
by
using
A'0°/
-
10
and
a
tetrameric
Mr
of
171000
(Callens
et
al.,
1985).
The
enzyme
concentrations
were
expressed
as
fM
active
sites,
and
the
enzyme
activity
was
monitored
by
the
coupled
D-xylose
isomerase/D-sorbitol
dehydro-
genase
assay
as described
by
Callens
et
al.
(1986)
and
Kersters-Hilderson
et
al.
(1987).
Metal-free
enzyme
was
prepared
as
previously
reported
(Callens
et
al.,
1986).
After
EDTA
treatment,
the
enzyme
was
analysed
for
residual
metal
contamination
by
atomic-absorption
spectrometry.
Only
0.03
equiv.
of
Co2",
0.016
equiv.
of
Mn2+,
0.14
equiv.
of
Mg2"
and
0.05
equiv.
of
Ca2"/monomer
remained.
Furthermore,
the
activity
was
less
than
3
%
of
that
observed
in
the
presence
of
10
mM-Mg2".
All
buffers
were
treated
with
Chelex
100
and
stored
in
acid-washed
plastic
containers.
Equilibrium
binding
studies
Equilibrium
dialysis
was
carried
out
in
Perspex
half-
cells
(Myer
&
Schellmann,
1962)
separated
by
a
semi-
permeable
membrane
[16
mm
(I
in)
dialysis
tubing
from
A.
H.
Thomas,
Philadelphia,
PA,
U.S.A.].
Before
use
the
half-cells
and
tubing
were
treated
with
10
mM-EDTA
and
rinsed
extensively
with
double-distilled
water.
Equal
volumes
(200
,u)
of
enzyme
and
Co2"
ligands,
dissolved
in
0.15
M-NaCl/0.01
M-triethanolamine/HCl
buffer,
pH
8.0,
were
loaded
on
both
sides
of
the
membrane.
The
cells,
sandwiched
in
a
holder,
were
allowed
to
equilibrate
under
rotation
(100
rev./min)
in
a
thermostatically
controlled
(35
°C)
Gallenkamp
orbital
incubator.
After
an
equilibration
time
of
4
h
at
35
°C,
the
portions
were
withdrawn
from
each
half-cell
and
Co2"
concentrations
were
determined
as
described
above.
The
extent
of
Co2"
binding
was
calculated
from
the
difference
in
Co2"
concentration
in
protein-containing
and
protein-free
compartments.
The
data
were
analysed
by
Scatchard
(1949)
plots.
Difference
spectrophotometry
U.v.
difference
and
visible
absorption
spectra
were
obtained
with
a
Uvikon-810
double-beam
spectrophoto-
meter
with
two
thermostatically
controlled
(25
°C)
2
cm
x
0.437
cm
mixing
cuvettes
(Yankeelov,
1963).
Equal
volumes
(800
ll)
of
enzyme
and
metal
ion
solution
in
0.01
M-triethanolamine/HCl
buffer,
pH
8.0,
were
added
to
the
compartments
of
the
reference
and
sample
cuvette.
After
base-line
recording,
the
sample
cuvette
was
mixed
and
the
spectrum
was
scanned.
For
difference
absorption
titrations,
two
ordinary
1
cm
x
1
cm
x
4
cm
cuvettes
were
used.
The
titrations
were
performed
as
described
by
De
Boeck
et
al.
(1982).
For
u.v.
difference
absorption
titrations
both
cuvettes
were
filled
with
2022
,1
of
enzyme
solution.
After
base-
line
recording,
portions
of
the
metal
ion
solution
were
added
to
the
sample
cuvette,
whereas
an
equivalent
volume
of
buffer
was
added
to
the
reference
cuvette.
For
visible-absorption
titrations
the
sample
cuvette
was
filled
with
2022
,l
of
protein
solution
and
the
reference
cuvette
with
an
equal
volume
of
buffer.
After
base-line
recording,
both
cuvettes
were
titrated
with
the
same
metal
ion
solution.
Metal
analysis
Metal
concentrations
were
determined
by
atomic-
absorption
spectrometry.
A
Perkin-Elmer
model
503
atomic-absorption/flame
spectrometer
was
used
for
Mg.
Ca
was
analysed
with
a
graphite-furnace
Hitachi
model
180-70
Zeeman
atomic-absorption
spectrometer,
and
Co
and
Mn
were
analysed
with
a
graphite-furnace
Perkin-Elmer
model
3030
atomic-absorption
spectro-
meter
with
2H2
background
correction.
Concentrations
of
Co2+
ions
were
also
determined
by
a
modification
of
the
Nitroso
R
salt
method
with
disodium
1-nitroso-2-hydroxynaphthalene-3,
6-disulphonate
(Vogel,
1962).
The
standard
assay
mixture
contained
0.7
ml
of
1.73
M-sodium
acetate
and
0.2
ml
of
2
%
(w/v)
Nitroso
R
salt
in
double-distilled
water.
After
addition
of
the
Co2+
sample
(100
,1)
and
mixing,
the
colour
intensity
was
read
immediately
at
510
nm
in
a
thermo-
statically
controlled
(25
°C)
Vitatron
photometer,
and
the
Co2+
concentrations
were
calculated
by
using
a
calibration
factor
of
1490
+
14
M-'.
RESULTS
AND
DISCUSSION
Evidence
for
Co2"
binding
to
a
high-affinity
site
Metal-free
enzyme
was
incubated
for
4
h
with
various
amounts
of
Co2"
(Table
1)
or
for
different
periods
of
time
with
a
constant
concentration
of
Co2"
(Table
2).
Cobalt
concentrations
were
then
determined
with
Nitroso
R
salt
and
compared
with
a
similar
set
of
experiments
without
enzyme.
From
the
data
in
Tables
1
and
2
it
follows
that
approximately
n-I
Co2"
ions
react
with
the
Nitroso
R
salt.
This
suggests
a
direct
and
tight
binding
of
1
equiv.
of
Co2"/monomer.
Evidence
for
very
slow
dissociation
of
the
Co2`-
enzyme
complex
was
obtained
from
spectral
changes
(510
nm)
in
the
presence
of Nitroso
R
salt.
Fig.
1
illustrates
that,
immediately
after
mixing
the
enzyme
with
1
equiv.
of
Co2"/monomer,
only
small
amounts
of
free
Co2"
could
be
determined.
Long
incubation
periods
with
Nitroso
R
salt,
however,
finally
resulted
in
complete
reaction
of
the
initially
added
Co2"
with
Nitroso
R
salt.
1988
286

Metal
ion
binding
to
a
D-xylose
isomerase
Table
2.
Cobalt
measurements
with
Nitroso
R
salt:
different
incubation
periods
of
enzyme
with
a
constant
concentration
of
Co2+
0.08
0.04
D-Xylose
isomerase
(82.5
/,M
active
sites)
was
incubated
with
250
,M-CO2+
in
0.01
M-triethanolamine
buffer,
pH
8.0,
at
35
°C
for
different
periods
of
time.
Free
Co2,
Co2+
deficiency
Time
determined
(/LM)
(equiv./monomer)
0
min
1
min
10min
20
min
30
min
60
min
2h
6h
25
h
80
177
176
174
172
170
169
165
166
160
0.86
0.90
0.92
0.95
0.97
0.98
1.03
1.02
1.09
70
60
L
0
2
50
40
0
10
20
30
40
Time
(min)
Fig.
1.
Dissociation
of
Co2l-enzyme
complex
in
the
presence
of
Nitroso
R
salt
D-Xylose
isomerase
(77.7
,UM
active
sites)
was
incubated
with
75.0
,uM-Co2+
in
0.01
M-triethanolamine
buffer,
pH
8.0,
at
35
'C.
0
2
r
Fig.
2.
Equilibrium
binding
of
Co2l
to
D-xylose
isomerase
from
S.
violaceoruber
The
data
from
equilibrium-dialysis
experiments
at
35
°C
are
plotted
in
accordance
with
the
Scatchard
equation.
Equal
volumes
(200
,ul)
of
enzyme
(42
/SM
active
sites)
and
20-408
/M-Co24
in
0.15
M-NaCI/0.0I
M-triethanolamine
buffer,
pH
8.0,
were
loaded
on
both
sides
of
the
membrane.
The
amount
of
bound
Co2+
was
estimated
from
the
difference
in
Co2+
between
the
two
half-cells.
Wavelength
(nm)
Fig.
3.
U.v.
absorption
spectrum
of
D-xylose
isomerase
obtained
in
the
presence
of
Co24
ions
Conditions
were
as
follows:
enzyme
(18.5
,UM
active
sites)
and
0.5
mM-Co2+
in
0.01
M-triethanolamine
buffer,
pH
8.0,
at
29
'C.
The
inset
shows
the
absorbance
difference
A(A296-A292)
of
D-xylose
isomerase
(16.9
,UM)
versus
equiv.
of
Co2+/monomer
at
35
'C.
Linearization
of
this
slow
Co24-nitroso
complex-forma-
tion
yielded
a
dissociation
rate
constant
of
0.148
min-'.
Total
Co2"
could
also
be
determined
after
drastic
treatment
of
the
enzyme
with
SDS
at
100
°C,
concomitant
with
dissociation
into
subunits
(results
not
shown).
Equilibrium
binding
of
Co24
Further
quantitative
measurements
of
the
interaction
of
Co24
with
D-xylose
isomerase
were
made
by
equili-
brium
dialysis.
The
data,
plotted
in
accordance
with
the
Scatchard
equation
(Fig.
2),
indicate
two
binding
sites/
monomer.
Information
concerning
the
first
Co2"
site
could
not
be
obtained
since
systematic
deviations
(results
not
shown)
occurred
for
n
<
1.
Binding
of
Co24
to
this
site
is
probably
too
strong,
and
concentrations
of
remaining
free
Co24
are
too
low
for
accurate
determina-
tion
by
atomic-absorption
spectrometry.
Only
the
lower-
affinity
Co2"
site
could
be
adequately
characterized
as
having
an
association
constant
of
4.0
x
104
+
0.4
x
104
MW1.
Co2`-induced
u.v.
difference
spectra
D-Xylose
isomerase
undergoes
conformational
changes
on
binding
of
Co2"
ions,
as
shown
by
u.v.
difference
absorption
spectrometry
(Fig.
3).
The
difference
absorp-
tion
spectrum
between
Co2+-free
enzyme
and
Co2+-
containing
enzyme
exhibits
two
absorption
maxima
at
296
and
289
nm
and
one
absorption
minimum
at
292
nm,
indicating
a
conformational
change
affecting
the
environment
of
tryptophan.
The
difference
absorbance
change
between
the
maxi-
mum
at
296
nm
and
the
minimum
at
292
nm
is
a
reliable
parameter
for
monitoring
the
binding
of
Co24
(Fig.
3
Vol.
250
0
4
o2
0
10
20
Time
(min)
287
1
r

M.
Callens
and
others
(a)
(b)
0.003'
0
0.004
I
1
\.002
/
%\
0
0.002
-
0.001
n .
aI0
a
450
550
650
Wavelength
(nm)
0
2
4
6
8
10
Co2"
(equiv./monomer)
Fig.
4.
(a)
Visible
absorption
spectrum
of
Co2+
-containing
D-xylose
isomerase
and
(b)
plot
of
absorbance
change
at
585
nm
versus
equiv.
of
Co2+/monomer
Conditions
were
as
follows:
(a)
enzyme
(18.5
,LM
active
sites)
and
0.5
mM-Co2+
in
0.01
M-triethanolamine
buffer,
pH
8.0,
at
29
°C;
the
broken
line
is
the
spectrum
of
0.5
mM-Co2+
in
0.01
M-triethanolamine
buffer,
pH
8.0;
(b)
enzyme
(24
,tM
active
sites)
titration
with
Co2',
in
0.01
M-triethanolamine
buffer,
pH
8.0,
at
35
°C,
as
described
in
the
Experimental
section.
inset).
After
the
addition
of
1
equiv.
of
Co2"/monomer
the
change
in
absorbance
remains
constant
at
its
maximum.
Thus
it
is
due
to
filling
of
only
one
site/monomer.
Assuming
a
bimolecular
association,
E
+
Co2+
=
E[Co,-]
(E[Co,-]
and
E[Co,Co]
represent
enzyme-metal-ion
complexes
with
Co2"
on
first
site
and
Co2`
on
both
sites
respectively),
a
binding
constant
of
>
3.3
x
10'
M-1
can
be
calculated.
Visible
absorption
spectra
of
Co2"-enzyme
complexes
Addition
of
excess
Co2`
to
apoenzyme
induces
a
visible
difference
absorption
spectrum
of
the
Co2+-
containing
enzyme.
The
difference
absorption
spectrum
between
the
Co2"-enzyme
complex
and
free
Co2`
ions
in
0.01
M-triethanolamine
buffer,
pH
8.0,
is
shown
in
Fig.
4(a).
The
spectrum
of
free
Co2`
(broken
line),
exhibiting
a
structured
absorption
around
500
nm
with
low
intensity
(e
<
6.9
M-1
cm-'),
is
characteristic
for
octahedral
sym-
metry
(Cotton
&
Wilkinson,
1980).
In
contrast,
the
difference
spectrum
of
Co2+-containing
enzyme
exhibits
maxima
at
585
nm
(e
135
m-1
cm-1)
and
500
nm
(e
104m-1
cm'1)
and
a
minimum
at
550
nm.
These
spectral
features
are
not
compatible
with
octahedral
symmetry,
but
are
rather
compatible
with
penta-
or
tetra-co-ordinate
Co2`
complexes.
However,
the
exact
co-
ordination
geometry
remains
tentative,
since
tetrahedral
or
penta-co-ordinate
Co2`
complexes
are
not
easily
distinguished
by
visible
absorption
spectra.
Differen-
tiation
can
only
be
made
on
the
basis
of
subtle
intensity
considerations,
tetrahedral
chromophores
having
a
larger
intensity
of
electronic
absorption
than
do
penta-
co-ordinate
complexes
(Bertini
&
Luchinat,
1984).
A
plot
of
change
in
A58,
versus
equiv.
of
Co2+/
monomer
is
shown
in
Fig.
4(b).
Up
to
the
addition
of
1
equiv.
of
Co2`/monomer
no
spectral
changes
were
observed,
in
contrast
with
the
u.v.
difference
spectra,
where
changes
were
only
observed
up
to
the
addition
of
1
equiv.
of
Co2`/monomer.
This
points
to
two
non-
identical
Co2`
sites,
as
previously
demonstrated
by
equilibrium-dialysis
experiments.
Since
there
is
no
absor-
bance
change
between
mono-Co2+-enzyme
and
solvated
octahedral
Co2+,
the
same
co-ordination
geometry
has
to
be
ascribed
to
the
Co2+
liganded
on
the
first
site.
From
Fig.
4(b)
it
follows
that
addition
of
more
than
1
(and
up
to
10)
equiv.
of
Co2+/monomer
causes
an
increase
in
the
A585.
From
the
titration
curve
an
apparent
association
constant
near
105-104
M-1
could
be
estimated,
in
agree-
ment
with
the
value
obtained
from
equilibrium
dialysis
for
the
second,
lower-affinity,
Co2`
site.
As
a
general
conclusion
the
spectrometric
studies
of
Co2'-containing
enzyme
clearly
demonstrate
binding
of
Co2+
to
two
environmentally
different
sites.
The
first
bimolecular
association,
E
+
Co2+
=
E[Co,-],
produces
changes
in
the
u.v.
spectrum
of
the
enzyme,
and
yields
an
association
constant
K
)
3.3
x
106
M-1.
Co2'
bound
at
this
conformational
site
appears
to
have
octahedral
symmetry.
The
second
bimolecular
association,
E[Co,-]
+
Co2+
=
E[Co,Co],
produces
changes
in
the
visible
Co2+
spectra,
and
yields
an
association
constant
105-104
M-1.
Co2+
binding
to
this
second,
lower-affinity,
site
is
involved
in
the
catalytic
process,
as
shown
by
kinetic
studies
(M.
Callens,
unpublished
work),
and
appears
to
be
tetra-
or
penta-co-ordinate,
as
deduced
from
spectral
intensities.
Competitive
interactions
of
Co2+,
Mg2+
and
Ca2+
In
the
following
experiments
the
interactions
between
Mg2+,
the
most
activating
ion,
Co2+,
the
most
stabilizing
ion,
and
Ca2+,
an
ineffective
ion,
were
studied
(Callens
et
al.,
1986).
Spectrometric
studies
of
apoenzyme
with
Mg2+
and
Ca2+
also
resulted
in
a
modified
u.v.
absorption
spectrum
(Fig.
5).
The
conformational
changes
of
the
enzyme,
affecting
the
tryptophan
and
tyrosine
environment,
are
very
similar
to
what
has
been
observed
with
Co2+.
The
difference
absorption
spectrum
of
Mg2+-containing
enzyme
exhibits
two
maxima
at
296
and
289
nm
and
one
minimum
at
292
nm.
The
change
in
absorbance
between
296
and
292
nm
was
virtually
complete
after
the
addition
of
3
equiv.
of
Mg2+/monomer
and
a
binding
constant
of
1
x
105
M-1
was
calculated
(results
not
shown).
Addition
of
Ca2+
to
the
apoenzyme
resulted
in
a
difference
absorption
spectrum
with
maxima
at
296
and
287
nm
and
a
minimum
at
291
nm
(Fig.
5).
The
plot
of
spectral
increases
between
296
and
291
nm
versus
Ca2+
increases
up
to
the
addition
of
3
equiv.
of
Ca2+/monomer
and
yielded
an
association
constant
K
=
5
x
I05
M-1
(results
not
shown).
1988
288

Metal
ion
binding
to
a
D-xylose
isomerase
0.006
0.004
I
0.002
-
0
I
-0.002
I
-0.004
I-
-0.006
260
280
300
320
Wavelength
(nm)
340
Fig.
5.
U.v.
absorption
spectra
of
D-xylose
isomerase
obtained
in
the
presence
of
Mg2+
and
of
Ca2+
Conditions
were
as
follows:
enzyme
(1
5
/M
active
sites)
and
152
,UM-Mg2+
(----)
or
78.1
/SM-Ca2+
(
)
in
0.01
M-
triethanolamine
buffer,
pH
8.0,
at
35
'C.
0.2
>i
0.1
0
1
2
r
Fig.
6.
Equilibrium
binding
of
Co2"
to
D-xylose
isomerase
in
the
presence
of
10
mM-Mg2+
The
data
from
equilibrium-dialysis
experiments
at
35
°C
are
plotted
in
accordance
with
the
Scatchard
equation.
Equal
volumes
(200
,ul)
of
enzyme
solution
(34
,/M
active
sites)
and
15-500
uM-CO2+
in
the
presence
of
10
mM-Mg2+
in
0.01
M-triethanolamine
buffer,
pH
8.0,
were
loaded
on
both
sides
of
the
membrane.
The
amount
of
bound
Co2+
was
estimated
from
the
difference
in
Co2+
between
the
two
half-cells.
The
almost
identical
positions
and
shapes
of
the
difference
spectra
between
metal-free
enzyme
and
mono-
Co2`-,
mono-Mg2+-
and
mono-Ca2+-protein
states
seem
to
suggest
binding
at
the
same
conformational
site,
or
at
least
co-ordination
with
some
identical
ligands.
Interaction
of
metal-free
enzyme
with
Co2"
in
the
presence
of
Mga+
was
studied
by
equilibrium
dialysis
(Fig.
6).
The
data
are
consistent
with
a
biphasic
Scatchard
plot,
representative
for
two
different
and
independent
Co2"
sites.
In
contrast
with
Fig.
2
(absence
of
Mg2"),
no
systematic
deviations
occurred
for
n
<
1,
which
allowed
calculation
of
both
Kapp
values.
The
slopes
yielded
Kap
1=
2.5x
101m-
and
Kapp.2
=
2x
10
m-1.
These
findings,
and
the
fact
that
both
Kapp.
values
are
smaller
than
the
association
constants
for
the
two
Co2+
sites,
indicated
competition
between
Co2+
and
Mg2a
for
the
same
binding
sites.
Competition
between
Co2+,
Mg2+
and
Ca21
for
the
second,
lower-affinity,
site
has
been
confirmed
by
kinetic
experiments
(M.
Callens,
unpublished
work)
and
by
spectroscopic
displacement
of
the
Co2+
visible
absorption
spectrum.
Fig.
7
shows
the
effect
of
the
gradual
addition
of
Ca21
to
Co2+-loaded
enzyme,
which
resulted
in
total
displacement
of
Co2+.
Gradual
addition
of
Mg2+
gave
only
partial
substitution,
owing
to
low
association
of
Mg2+
with
this
site
(results
not
shown).
In
general,
the
competition
experiments
monitored
by
means
of
equilibrium
dialysis
and
spectrometric
displace-
ment
studies
provide
evidence
for
metal
ion
binding
at
the
same,
if
not
at
very
close,
binding
sites.
Vol.
250
0.001
0
K
-0.001
~1
-0.002
-0.003
400
500
600 700
Wavelength
(nm)
Fig.
7.
Visible difference
spectrum
of
Co2+-1oaded
D-xylose
isomerase
in
0.01
M-triethanolamine
buffer,
pH
8.0,
at
35
°C
Conditions
were
as
follows:
enzyme
(23
#uM
active
sites)
and
237
/SM-CO2+
in
the
sample
cuvette,
and
237
,uM-Co2+
in
buffer
in
the
reference
cuvette.
Spectrum
a,
base-line
recording
with
visible
Co2+
difference
absorption
spectrum
in
memory;
spectrum
b,
displacement
of
Co2+
spectrum
after
addition
of
73
#M-Ca2+;
spectrum
c,
displacement
of
Co2+
spectrum
after
addition
of
237
1tM-Ca2+;
spectrum
d,
displacement
of
Co2+
spectrum
after
addition
of
658
/M-
Ca2+.
a
b
d
.
..
289

290
M.
Callens
and
others
We
thank
M.
Cambier
and
B.
Desmet
for
technical
assistance,
and
Dr.
F.
G.
Loontiens
for
spectrophotometric
facilities
made
possible
by
a
research
grant
from
the
National
Fund
for
Scientific
Research.
R.
C.
is
a
Senior
Research
Associate
of
the
National
Fund
for
Scientific
Research.
M.
C.,
P.
T.
and
W.V.
are
bursars
of
I.W.O.N.L.
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Received
5
June
1987/10
August
1987;
accepted
27
October
1987
1988