Vol. 67, No. 4, 1975
BIOCHEMICAL
THE CATALYTIC
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
METAL ATOMS OF COBALT SCSSTITUTED
LIVER ALCOHOL DEHYDROGENASE Arthur
J. Sytkowski*and
Bert
L. Vallee
Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, Division of Medical Biology, Peter Bent Brigham Hospital, Boston, !Yass.
Received
November
I,1975
SUMMARY: The catalytic and non-catalytic Zn atom pairs of horse liver alcohol e dro enase (LADH) have been replaced sequentially either by 65Zn, Co or E-2 co. The Co derivatives exhibit characteristic spectra. When Co replaces the Zn atoms which exchange secondly, enzymatic activity is altered, and both imidazole and l,lO-phenanthroline (OP) significantly modify the spectrum of the catalytic Co atoms. Further, due to the removal of cobalt, the instantaneous and reversible OP inhibition of the native enzyme becomes t!me-dependent and irreversible. Jointly, these data identify the pair of metal atoms of LADH which exchange secondly under the present conditions as the catalytic one. The approach described provides a basis for the differentiation of catalytic and non-catalytic metal atoms of multichain metalloenzymes. INTRODUCTION:
The four
have been classified and replacement exhibit
but one pair spectrally in the
X-ray
catalytic
have
activities
in which of either
structure
analysis
(3-6)
(2).
cne or both
Attempts
can be shown to correspond
to that
pairs
(l),
which
to replace
the metal
of site-specific
pairs
(LADH)
enzyme species
We have now prepared
Zn by Co atoms.
of crystals
or structural,
to differentiate due to lack
either
dehydrogenase
respective
of each
Co in order
Co replaces pair
the
characteristic
of the
alcohol
and non-catalytic,
been inconclusive
replacement
liver
by Co or Cd yields
of Zn atoms with
derivatives location
into
of both
catalytic
Zn atoms of horse
atom pairs conditions
Co-LADH
of Zn atoms,and now known
from
the the
(7).
METHODS: LADH (Boehringer-Mannheim) containing 4 g-atoms Zn/mole enzyme was .-labeled with 65Zn by exchange experiments so that pairs of intrinsic atoms are sequentially replaced to yield [(LADH)65Zn2Zn2] or [(LADH)Zn266Zn2] (8), where the order of the atoms indicates the sequence of replacements thereby providing site-specific monitors for subsequent quantitative replacement with Enzymatic activity, metal content, and radioactivity were determined and LADH was dialyzed against CoCl2 Reagents were purified as described (8). *Fellow
of the Helen
tlay Whitney
Foundation
Vol. 67, No. 4, 1975
(Johnson-Matthey) Absorption spectra l,lO-Phenanthroline and imidazole from
BIOCHEMICAL
by a modification of the method of Drum and Vallee (2). were obtained with a Cary 14 recording spectrophotometer. (OP) was obtained from G. Frederick Smith Chem. Coro. Sigma Chem. Co.
RESULTS __----
AND DISCUSSION:
species
in
zinc
which
atoms
content are
and all
enzyme
is
and,
Co the
[(LADH)Co2
second,
of
I).
(Table
in
enzymatic
activity
that
the Both
at
655
~74~ in
(750)
analysis
for
of of
the of The
Co atoms.
geometry
reflect of
ligands
in
absorption both
the
Zn
the
enzymes upon
65Zn
of
from
the
of
[(LADtl)65Zn2Zn2]
[(LAD11)Co2Zn2] 2 g-atom
to
Upon
that
Co
of
the
the
second
Co/mole
and
I),
a bsorb
visible
1.9
Zn
enzyme
pair
LADH,
(Table
and
of
but
g-atom
Zn
atoms
its
a first
suggestion
one.
for
of two
x655
bands.
in
The
(7).
the
coordination
spectra
of
are
These
the
apparent
1489
mixed of
the
and
coordination ion
(9)
crystallographic of
chromophoric,
metal
>
their
geometry
examination
the
(1050)
a difference
maxima
X-ray
permit of
A655
tetrahedral
from
spectra
(c)
revealing
these
a distorted
resultant
ligands.
of
radiation
of
(75D),
position
sphere
properties
Co2Zn2]
h740
with
LADH
enzyme
[(LADli
(2050),
Co complexes
chromophoric
of
65Zn
in
mg-'
absorptivities
of
the
g-atom
= 9 min-1
the
Co replacement.
equal
maximally
geometries
native
340
zinc
exchange
loss
replaces
3.9
total
Importantly,
contains
Co then
the
intramolecular
resulting
is
intrinsic
and
4".
i.e.,
[(LADti)Co2C02]
these
those such
Zn atoms,
of
identical
site-specific
contains
[(LAD~!)CO~CO~] for
or
Zn2],
catalytic
molar
resemble
substitution
should
the
are
enzyme
pairs
Both
Co incorporation
o-f
I\A
second
pH 7,
inter-
against
and
and
at
activity
which
is
I)
similarly
specific
decreased,
nm with
existence
either
the
the
enzyme.
[(LADH)Co2Zn2]
its
is
or
stored
;(L,4DH)Zn,65
[(LADH)co~CO$
absorbance
intensities The
of
properties,
(Table
pair
dialysis
740
native
for
first
[(LADlH)Co2Zn2]
and
molar
and
pair
the
when
respectively.
second
from
of
Continued
resulting
first
parameter
65Zn
enzyme,
the
subsequent
the
isotopic
for
during
replaces
@jZn2],
Zn/mole
weeks
no evidence
hence,
their
activity
several
a reliable
dialysis,
but not
enzymatic
there
is
for
substitutes
specific for
65 Zn
Except
indistinguishable
stable
storage, Zn f
65Zn
are
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
catalytic complexes
and
the
nature
and
Vol. 67, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I PROPERTIES OF LIVER ALCOHOL DEHYDROGENASES Me/LADH g-atom/mole ~65Zn
4
-
-
14
4
2
-
14
4
2
-
14
[(LADH)Co2Zn2]
1.9
0
2
14
[(LADH)Co,G"Zn,]
1.9
1.9
2
14
[(LADH)CO~CO~]
co.2
co.1
3.9
9
[(LADH)Zn265Zn2]
Imidazole
is
known
water
and forming
alter
the absorption
[(LADH)Co2C02]
to interact
with
an LADH.Zn.imidazole spectrum
shifting
absorptivity
its
mixed
while
generating
interacts
second
the
665 and 634-640
nm bands,
support
view,
of this The striking
the
are
absorption
L(LADH)Co2C02], Functionally, as instantaneous,
this
agent
with also
but
in effect
rendering
OP binding reversible
it
the
this
does not
data
suggest
(Fig. identical
of the
1490
by the
ones.
In
of [(LADH)Co2Zn2].
(OP),
a chelating
agent
Zn atoms of the native OP does not
to that
enzyme
1B).
imidazole
spectrally
1A) but markedly
Zn atoms
of
nm (Fig.
that
the spectrum
catalytic
that its
at 634-640
interpretation.
to the catalytic inhibition
Imidazole
1A) but perturbs
of l,lI)-ohenanthroline
of [(LADH)Co2Zn2J
by displacing
atoms as the catalytic
does not affect
with
atoms
now characterized
not to remove
in agreement
spectrum
These
these
effects
(7).
a shoulder
of Co atoms,
identifying
spectral
known to coordinate (10)
pair
(Fig.
metal
655 to 665 nm and increasing
the maximum at 740 nm.
with
complex
of [(LADH)Co2Zn2] maximum from
(~=2300)
__Co
the catalytic
It does not affect
enzyme
min -1 mg -1
Zn [(LADH)Zn2Zn2]
molar
*A340
alters
of [(LADH)Co2Zn2] of native
(lc)),
change that
of (Fig.
1C)
LADt( manifests
reflecting
the formation
BIOCHEMICAL
Vol. 67, No.4,1975
Figure 1: or presence Absorption imidazole. or presence or 1 mM DP:
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Panel A: Absorption spectrum of [(LADH)Co2Zn2] in the absence (-) Panel B: of either imidazole or l,lO-phenanthroline (----). spectrum of [(LADH)CO~CO~] in absence (-) or presence (----) of Panel C: Absorption spectrum of [(LADH Co2Co2] in absence (-) 8 x lo- !! M enzyme: 50 mM imidazole (----) of l,lO-phenanthroline. 0.2 M Tris-acetate, pH 7.5, 4'.
Lti
1
/
2
4
6
MINUTES
Figure 2: The inhibition of [(LADlI)Co2Zn2] and [(LADH)Co2Co2] by l,lO-phenanthroline. 10 119 of [(LADH)Co2Zn2] is added to a cuvette containing 3 ml of 1 mM OP in the assay solution. Inhibition is observed "instantaneously" (lapsed time = 8 set) and does not change with time (----). Addition of Cu2+ or Zn2+ (3 mM final concentration) during the 120-Fold assay (left arrow) increases the activity within 10 sec. dilution of the solution containing enzyme and OP reverses the inhibition completely (right arrow). [(LADH)Co2Co2], 1.2 x 10-5 M, is incubated with 7 x 10-5 M OP, 0.2 M Tris-acetate, pH 7.5, 0" diluted 120-fold and assayed in the absence of OP ( -). Inhibition is time-dependent and not reversed either by addition of excess metal ions (solid curve, X) or further dilution (solid curve, XX). of a 1:l interacts
enzyme.OP with
time-dependent:
complex:
other
LADHsZn + OP 1 LADH.Zn.(OP)l.
metalloenzymes
to remove
E-Me + nOP z E + Me(OP),,
Zn,the
and for
1491
In contrast,
accompanying
LADH it
is
inhibition
irreversible.
when OP is Hence,
Vol.67,No.4,1975
it
would
would
be expected
dependent,
but
and analytical
exchanges
pair
from
of excess This
the second
pair
Further
proof
second
catalytic
[(LADH)Co2Co2] properties
to that
of the
Co sulfur
complexes than
conversely,
LADH, as is
lower
than
analogous
with
OP removes
Co spectrum
than
of
the thermodynamic The stabilities
of
site,
could
of the corresponding
complexes
to remove Co more readily
that
the
of [COG]
[Zn(OP)n)
pair.
at the catalytic
those
the stabilities
of
by OP to that
showing
systems.
such as exist
dilution
inhibition
converting
model
differs
Neither
catalytic
data
consistent
from
it
both sets of data suggest
from
thereby
[(LADH)Zn2Zn2
Zn
complexes (11).
Zn from
are much
On this the
basis,
active
site
of LADIi constitutes
a
of
observed.
Replacement chemical
of the
be expected
derives
conditions,
the OP
affected
enzyme to be the
as deduced complexes,
atoms
Thus,
of the
to be significantly
those
of metal
the
the
identify
native
Zn enzyme.
reverses
and of
present
both
there-
with
In contrast,
native
metals pair
the
and irreversible;
of the
the
We have,
definitively
inhibits
is time-dependent
of Co atoms
system
while,
under
of [(LADH)Co2Zn2],
and nitrogen
be expected
which,
hypothesis
pair
not.
Together
as that
Co
to time
[(LADH)co~Z~~]
they
and reversibly.
atoms
of this
would
lA-C),
exchangessecondly.
of metal
pair
l,lO-Phenanthroline
localizes
of Zn atoms with
instantaneous
Figs.
Zn or of other
of Co atoms which
LADII, from
I,
instantaneously
[(LADH)CO~CO~J.
OP would
(Table
2).
pair
the above expectation.
out
the mode of inhibition
nor addition
of Zn with
modification
the functional the
data
of [(LADH)CO~CO~]
markedly
higher
bear
(Fig.
and [(LADH)Co2Zn21
catalytic
of [(LADH)Zn2Zn2],
of [(LADH)Co2Co2]
secondly
inhibition
OP inhibits
the mode of inhibition
the catalytic
this
of the
of the non-catalytic
[ ( LADH)CO~CO~]. The data
pair
replacement
replacement
examined
spectral
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the mode by which
alter
fore,
that
BIOCHEMICAL
under
characteristics
instantaneous
inhibition
of the Co enzyme,to
our
Co at the catalytic very
mild
conditions
of the of the
knowledge,
site
resultant
Zn enzyme is
thus
far
1492
but which species. into
drastically The conversion
a time-dependent
unique
alters
in enzymes
of
inhibition having
under-
Vol. 67, No. 4, 1975
gone metal-assisted
BIOCHEMICAL
dechelation.
may prove
applicable
in other,
similar
to localize
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The combinations metal
of procedures
atoms and their
functional
here
employed
potential
systems.
Acknowledgements __This
work
was supported
by Grant-in-Aid
GM-15003.
References 1. 2. 3. 4. Z: 7. 8. 9.
10. 11.
Drum, D.E., Harrison, IV, J.H., Li, T.-K., Bethune, J.L. and Vallee, B.L. (1967), Proc. Nat. Acad. Sci. USA 5i-, 1434-1440. Drum, D..E. and Vallee, B.L. (1970), Biochem. Biophys. Res. Commun. 42, 33-39. Young, J.FI. and Wang, J.H. (1971), J. Biol. Chem. --246, 2815-2821. Takahashi, M. and Harvey, R.A. (1973), Biochemistr 12, 4743-4750. Drott, !H.R., Santiago, 0. and Shore, J.D.-bEF (1974 , Let!. 39 21-23. Sloan, D.L., Young, J.Pl. and Mildvan, A.S. (1975),-B,ochem,stG'14 .____ - --I 1998-2008. Br$nd&n, C.-I., Jornvall, Ii., Eklund, H. and Furugren, E.,The Enzymes, in press. Drum, D.E. , Li, T.-K. and Vallee, B.L. (1969), Biochemistry 8, 3792-3797. H.A.O. and i!illiams, R.J.P.-.(1970), in Chemical Foster, M.A., I-lill, Reactivity and Biological Role of Functional Groups in Enzymes, Biochemical Sot. Symp. No. 31, Smellie, R.M.S., Ed., London, Academic Press, p. 187-202. Vallee, B.L. and Hoch, F.L. (1957), J. Biol. Chem. 3, 185-195. Sill&, L.G. and Flartell, A.E. (1964-r-Stabmy-Constants of !letal -Ion Complexes, Spec. Publ. 17. --Chem. Sot. (London),
1493