S Yvert Sen 1984
S Yvert Sen 1984
S Yvert Sen 1984
SYVERTSEN
Institute,
University
AND
of
JOHN S. MCKINLEY-MCKEE
Oslo, P.O. Bm
1041, Blindem,
Oslo 3, Norway
The affinity of nitrogen and sulfur ligands for the catalytic zinc ion in horse liver
alcohol dehydrogenase has been investigated by their influence on the affinity labeling
reaction with iodoacetate. All the nitrogen compounds including ammonia, a primary
and a secondary amine, and heterocycles containing a pyridine-type
nitrogen with the
exception of 2,2-dipyridyl were found to activate the affinity labeling reaction. Activation
results from inner-sphere ligand coordination to the catalytic zinc ion. Closely related
pyridine compounds gave a regular increase in affinity for the enzyme with increasing
basicity, as expected for coordination to a metal ion. The sulfur compounds penicillamine
and mercaptoethanol also activated the affinity labeling reaction, but dimercaptopropanol
bound very tightly as a bidentate inhibited the reaction. The anions hydrosulfide, diethyldithiocarbamate,
and cyanide coordinated to the catalytic zinc ion, whereas azide,
thiocyanate, tetrazole, and iodide complexed the anion-binding
site. The anionic metal
ligands increased the rate of inactivation
of the enzyme with iodoacetamide by binding
to the catalytic zinc ion, while the binding of iodoacetate to the anion-binding
site was
prevented.
The catalytic zinc ion in horse liver alcohol dehydrogenase is liganded by three
protein ligands, while at least one position
in the coordination sphere is occupied by
a solvent-exchangeable
water molecule (1).
The enzyme is inactivated when the protein
ligand Cys-46 is alkylated by iodoacetate,
iodoacetamide,
or a-bromo+imidazolyl
propionic acid. Iodoacetate and cy-bromofl-imidazolyl
propionic acid react via an
affinity labeling mechanism, whereas iodoacetamide reacts without forming any
detectable reversible complex with the enzyme and is less specific for Cys-46(2-4).
In the reversible complex with the enzyme,
iodoacetate is bound to the anion-binding
site of which Arg-47 is a part; cY-bromo+
imidazolyl propionic acid also binds to the
catalytic zinc ion.
Some neutral ligands like imidazole,
1 To whom correspondence
l,lO-orthophenanthroline,
and 2,2-dipyridyl coordinate the catalytic zinc ion (5).
Imidazole has been shown to activate the
affinity labeling reaction with iodoacetate
and the inactivation
with iodoacetamide.
This resulted from the electronic properties of imidazole as a metal ligand (6). In
the present work a number of nitrogen and
sulfur ligands have been investigated
to
compare their preference and affinity as
ligands for the catalytic zinc ion.
The anion-binding
site and the catalytic
zinc ion are essential for coenzyme and
substrate binding. The two binding sites
are close in space and there are positive
or negative cooperative
interactions
in
binding to these sites (7,8). Cooperativity
is seen in the decrease in affinity for anions
to the anion-binding
site associated with
the ionization of the zinc-bound water. This
cooperativity has been studied in this work
by using anions such as cyanide, hydrosulfide, and diethyldithiocarbamate,
which
should be addressed.
159
0003-9861/84 $3.00
Copyright
All rights
160
SYVERTSEN
AND
AND
METHODS
Reagents. The enzyme horse liver alcohol dehydrogenase (EC 1.1.1.1.) was from Boehringer-Mannheim.
The purity and assay were as previously described
(2). Before use the enzyme was dialyzed against three
changes of a lOO-fold vol of 30 mM Hepps/NaOH
buffer, pH 8.2. The buffers Mes, Tes, and Hepps were
obtained as free acids from Sigma. Borax, 2,2-dipyridyl, and t-butanol were from Merck. Acridine orange
(3,6-dimethylaminoacridinium
chloride)
was from
Serva and CoClz. 6HaO from Riedel-de Haen. Iodoacetic acid and iodoacetamide were from Sigma. Iodoacetic acid was recrystallized
twice from petroleum
ether and iodoacetamide
from water. All other reagents were the purest commercially
obtainable.
Double quartz-distilled
water was used for all solutions.
Inactivations.
Enzyme inactivations
were carried
out at 23.5C using an enzyme concentration
of about
5 pM. For inactivations
with iodoacetamide 10 mM of
reagent was used. Buffer concentrations
were 20 mM
Mes at pH 6.1, 20 mM Hepps at pH 8.2, and 20 mM
borax at pH 10.0.
Data processing. Linear first-order
plots of log(activity) against time were treated by linear regression
analysis. The data obtained for the observed firstorder rate constant, /cobs, as a function of the concentration
of iodoacetate and one or two effecters
were treated by the derivative-free
nonlinear regression program BMDPAR (copyright 1981, University
of California,
Los Angeles). Computing was carried
out on the DEC-10 machine of the University of Oslo.
Cobalt(II)-substituted
enzyme. The catalytic zinc ion
was selectively removed by dialysis (9). Insertion of
Co(I1) ions was performed with dissolved enzyme apohybride in 25 mM Tes, pH 8.2. A 1.5-fold excess of
CoCl* * 6HaO in relation to the concentration
of subunits was used. Absorption spectra were recorded with
a Hewlett-Packard
8450A spectrophotometer.
The
concentration
of apo-hybride was determined using
the molar absorptivity
of e280= 36000 M-l cm- (9),
based on M, = 80,000 (10).
2,2-dimridyl
as an optical probe. The binding of 2,2-
* Abbreviations
used: Mes, I-morpholineethanesulfonic
acid; Tes, 2-{[2-hydroxy-l.l-bis(hydroxymethyl)ethyl]amino}ethanesulfonic
acid; Hepps, 4-(2hydroxyethyl)-1-piperazinepropanesulfonic
acid.
MC KINLEY-MC
KEE
derived
2E&t + 2.WGpp
+ ~PJG,,I,
PI
from
MW
A = e.l.[EdP],
Et = [E] + [EdP],
Kspp = and
[EdP]
KL = [Ll/((&/s) - 1).
PI
Scheme1
K, is the dissociation constant of the enzyme-inactivator complex, and b is the maximum first-order
rate constant for inactivation.
With excess inactivator(1) a pseudo-first-order
reaction is observed with
respect to active enzyme, for which the observed firstorder rate constant is given by
kbs
MWVG + [II).
[31
ZINC
BINDING
IN HORSE
LIVER
ALCOHOL
DEHYDROGENASE
k2
EI - E(inactive)
161
mixed-type inhibitor
with respect to iodoacetate. All the other nitrogen ligands
+
+
were activators of the affinity labeling reaction. Typical examples of double-inverse
plots in the presence of an activator are
K i
q
shown for thiazole at pH 8.2 in Fig. 2 and
EL + I-& EIL az E(inactive)
for cyclohexylamine
at pH 10.0 in Fig. 3.
Scheme II
Activation is demonstrated by the ordinate
intercept in the presence of activator being
Here KL is the dissociation constant of the enzymebelow the intercept without activator. This
effector complex, a is an interaction or cooperativity
is reflected by the parameter @> 1 (Scheme
constant, and fl is a constant affecting the maximum
II). Figures 2 and 3 differ regarding the
rate constant. For scheme II kob is expressed by
pattern of the intercepts on the abcissa.
k,[I](l + flL]/aKd
This is a consequence of an altered interkobs=
t41
KIU + IL]/&) + Ul(1 + [Ll/aK,)
action constant cy. In Fig. 2 (Y > 1 means
Loading the BMDPAR program with this function
there is negative cooperativity in the bindalong with the data for the dependent variable kobs ing of iodoacetate and thiazole to the enand the appropriate variables [I] and [L], allows dezyme at pH 8.2, while in Fig. 3 CY< 1 demtermination
of the parameters (Y, 8, and KL; KI and
onstrates positive cooperativity
in the
b were determined previously (13).
binding of iodoacetate and cyclohexylamine to the enzyme at pH 10.0. Table I lists
RESULTS
the dissociation constants KL and the paThe e#ect of sme neutral nitrogen and rameters (Y and p according to Scheme II
sulfur ligands. The influence of effector li- for the nitrogen and sulfur ligands exgands upon affinity labeling with iodoacamined.
For the activators cyclohexylamine,
pietate is illustrated by double-inverse plots
of kobsversus iodoacetate concentration at peridine, and 4-dimethyl-amino
pyridine,
different effector concentrations.
Figure 1 the experiments were performed at pH 10.0
to ensure some of the effector being in the
is such a plot at pH 8.2 with the effector
ligand 2,2-dipyridyl,
which is a partial
base form.
E+I2
[2.2- Dipyridyl]
- 0.4
-a2
0.2
a6
I/ Dodoacetate]
FIG. 1. Double-inverse
plot of the observed first-order
concentration
for the inactivation
of horse liver alcohol
Buffer, 20 mM Hepps, pH 8.2.
(mbl)
1.0
(mMr
rate constant,
dehydrogenase.
162
SYVERTSEN
AND
MC KINLEY-MC
[Thiazole]
-0.2
KEE
(mM)
Q6
0.2
I/ [lodoacetate]
FIG. 2. Effect of thizole.
Buffer,
20 mM Hepps, pH 8.2.
[Cyclohexylamine]
l/ [lodoacetate]
FIG. 3. Effect of cyclohexylamine.
1.0
(mM)-
Buffer,
(mM)
(mM)-
20 mM borax, pH 10.0.
ZINC
BINDING
IN HORSE
LIVER
EL2 +Lz
+
E+l
163
DEHYDROGENASE
EI?
E(inactive)
Ll
-&.I
ALCOHOL
Ll
KL1
J
ELILB s
Scheme III
This is an extension of scheme II where KL2 is the dissociation constant for the second
effector from the enzyme and y is an interaction constant. Equation [5] expresses kobs
as a function of [I], [L,], and [L,]:
k
&JI](l + ,&]/aKd
Ohs
= KAl + M/&I
+ LI/KLz + MLI~Y&JL)
+ [IN1 + LI/~Kd
[51
tion constant of the effector ligands, calculated from Eq. [2]. For cyanide a plot of
absorbance against the concentration of
2,2-dipyridyl deviated from the other plots
in that the initial part of the curve was
sigmoidal. This resulted in higher standard
deviations in the results. 2,2-dipyridyl is
displaced from the enzyme by cyanide, hydrosulfide, diethyldithiocarbamate,
and
dimercaptopropanol as indicated by the
The inJEuence of eflector ligands on the values of KaPP. Except for cyanate (see Disbinding of .Z,.%dipyridyl. Table III lists KaPP cussion) there is good agreement between
and c307
for the binding of 2,2-dipyridyl cal- the values of KL in Tables II and III.
culated from Eq. [l] and KL, the dissociaInactivation
of the enzyme by iodoacet-
164
SYVERTSEN
AND
MC KINLEY-MC
TABLE
KEE
BINDINGOFNITROGEN-AND SULFUR-CONTAINING
LIGANDSTOHORSE LIVER ALCOHOL
DEHYDROGENASE
ACCORDINGTO SCHEMEII
Effector ligand
2,2-Dipyridyl
4-dimethylamino-pyridine
I-Methylpyridine
Pyridine
3-Chloropyridine
I-Cyanopyridine
Pyrazine
Imidazole
Imidazole
N-Methylimidazole
Thiazole
Acridine orange
Cyclohexylamine
Piperidine
Ammonia
Hydrogen sulfide
Mercaptoethanol
Penicillamine
Dimercaptopropanol
PH
P&
KL* 6-Q
8.2
4.44
0.55 + 0.1
0.61 + 0.13
10.0
10.15
0.71 rt 0.05
8.2
8.2
8.2
8.2
8.2
8.2
6.08
5.21
3.07
0.32 + 0.02
2.5 3~ 0.3
1.5 r 0.2
0.36
2.2
1.7
1.5
1.95
9.1 k 2.6
10.0
8.2
8.2
8.2
0.6
7.00
7.00
7.06
2.53
10.0
10.0
10.0
10.1
10.6
11.0
9.26
8.2
7.04
6.1
6.1
6.1
9.43
-
67
1.4
5.6
0.24
11
0.06
rt 13
+ 0.1
+ 0.1
* 0.02
+ 0.8
+ 0.02
1.8 k 0.1
0.83 5 0.1
220 2 33
0.07 + 0.01
1.2 f 0.1
32 k 13
3 f 1 (PM)
+
+
+
+
0.07
0.2
0.3
0.2
1.7 k 0.3
4.5 + 1.5
2.4 f 0.3
0.47 + 0.02
1.2 -c 0.2
2.0 + 0.3
0.56 f 0.07
0.34 zk 0.04
0.39 + 0.04
0.93 + 0.3
60 + 6
4.2 5 0.8
4+1
4.3 f 0.8
0.10 + 0.03
4.6
2.7
3.6
2.5
k 0.3
+ 0.1
k 0.3
+ 0.1
1.7 k 0.2
5.4 f 1.7
6.8 5 0.4
5.3 + 0.1
4.8 31 0.3
5.9 * 0.5
1.2 t- 0.1
5.4 + 0.3
1.4 f 0.1
9.4 -c 3.2
6+3
1.4 + 0.1
10 (est.)
0.9 32 0.07
aK, is the proton dissociation constant. For the nitrogen bases it refers to the conjugated acid (14, 15).
* KL is the dissociation constant for the ligands from the enzyme.
cxis the interaction constant for simultaneous binding of iodoacetate and ligand to the enzyme.
d/I is a constant affecting the maximum rate of inactivation.
ePrevious determination (8).
Activation of a@@ labeling with iodoacetate. The effect of the ligands in Table
ZINC
-0.2
BINDING
0.2
l! [lodoacetate]
IN HORSE
06
LIVER
1.0
(rnM~
I on the affinity labeling reaction with iodoacetate results from their coordination
to the catalytic zinc ion of liver alcohol
dehydrogenase. For imidazole the observed
activation has been considered as an innersphere ligand effect, where acting as a
metal ligand, imidazole is a better electron
donor than water (6,16). Substituting
for
water, imidazole increases the electron
density on the metal and on Cys-46, which
becomes a better nucleophile and more reactive. This is supported by the present
results as all the monodentate nitrogen and
sulfur ligands show the same activation
pattern as imidazole. It also correlates with
the better donor properties of sulfur and
nitrogen compared to oxygen.
Unlike the other nitrogen compounds,
2,2-dipyridyl does not stimulate affinity labeling but is a mixed-type inhibitor.
For
2,2-dipyridyl
at pH 8.2 (Y < 1 and fi $ 1
which is the opposite of that found for all
the nitrogen compounds other than l,lOphenanthroline
(8). The low reactivity of
the ternary enzyme-2,2-dipyridyl-iodoacetate complex (p 4 1) indicates that steric
effects of the large bidentate ligand can
make the access of iodoacetate to Cys-46
difficult. CY< 1 means that 2,2-dipyridyl
facilitates the binding of iodoacetate to the
enzyme.
ALCOHOL
165
DEHYDROGENASE
II
ANION BINDING TO HORSE LIVER ALCOHOL DEHYDROGENASE IN THE ABSENCE AND PRESENCE OF
IMIDAZOLE ACCORDING TO SCHEME III
Anion, Lz
Azide
Iodide
Tetrazole
Thiocyanate
Cyanate
Diethyldithiocarbamate
Cyanide
Decanoate
Decanoate
PH
KI.z (mM)
8.2
8.2
8.2
8.2
8.2
3.7
3.6
3.7
1.3
0.89
f
+
f
f
f
0.5
0.5
0.5
0.1
0.07
4.2 + 0.8
2.0 rf: 0.5
6.5 f 2.1
1.9 f 0.2
24+9
8.2
8.2
8.2
10.0
1.1
0.24
0.050
0.58
f
f
f
f
0.1
0.02
0.005
0.12
a,
03
>150
co
166
SYVERTSEN
AND
MC KINLEY-MC
TABLE
KEE
III
TITRATIONOFALCOHOLDEHYDROGENASEWITH~,~-DIPYRID~INTHEPRESENCE
OFEFFECTORLIGANDSAT pH 8.2
Effector
Molar absorptivity
[em7 (lo4 M-' Cm-')]
ligand
Dimercaptopropanol
(6 PM)
Hydrosuhide
(170 PM)
Cyanide (0.6 mM)
Diethyldithiocarbamate
(3.5 mM)
Cyanate (6.7 mM)
Thiocyanate (4.0 mM)
Azide (12 mM)
Tetrazole (12 mM)
Kw;
1.71 + 0.06
1.33 + 0.05
1400 + 110
2.6 f 0.03
1.3 f 0.1
1880 f 400
1723 f 250
1080 f 70
1.1 f 0.03
763 f
227 f
480 f
460 +
400+15
2.26 + 0.01
2.04 k 0.03
2.01 f 0.06
hb (PM)
(PM)
1.79 f 0.02
3.3
67
262
900
4300
43
3
20
35
b I
1.0
[Effector]
I I
2.0
#J
(rnbl)
FIG. 5. Inactivation
of horse liver alcohol dehydrogenase by iodoacetamide. The second-order rate constant, k, for inactivation
versus the concentration
of
(0) hydrosulfide; (0) cyanide; and (Cl) diethyldithiocarbamate. Buffer, 20 mM Hepps, pH 8.2.
460
560
700 "In
ZINC
BINDING
IN HORSE
LIVER
ALCOHOL
DEHYDROGENASE
167
168
SYVERTSEN
AND
MC
KINLEY-MC
KEE
REFERENCES
1. BRHNDCN, C. I., JBRNVALL,
H., EKLUND, H., AND
FURUGREN, B. (1975) in The Enzymes
(Boyer,
P. D., ed.), 3rd ed., Vol. 11, pp. 104-190,
Academic Press, New York/London.
2. REYNOLDS, C. H., AND MCKINLEY-MCKEE,
J. S.
(1969) Eur. J. Bioch~wz. 10,474-478.
3. DAHL, K. H., AND MCKINLEY-MCKEE,
J. S. (1977)
Eur. J. Biochem. 81,223-235.
4. EVANS, N., ANDRABIN, B. R. (1968) Eur. J. Biochem
4.548-554.
5. BOIWE, T., AND BRHND~N, C. I. (1977) Eur. J.
Biochem 77,173-179.
J. S. (1981)
6. DAHL, K. H., AND MCKINLEY-MCKEE,
Eur. J. Biochem 120,451-459.
ANDERSSON,
P., KVASSMAN,
J., LINDSTR~M,
A.,
OLDEN, B., AND PETTERSSON, G. (1980) Eur. J.
B&hem. 108,303-312.
SWERTSEN, C., AND MCKINLEY-MCKEE,
J. S. (1983)
Arch
Biochem. Biophys.
223,213-223.
ZINC
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
BINDING
IN HORSE
LIVER
ALCOHOL
DEHYDROGENASE
169
J.
of