Vol. 87, No. 1, 1979
8lOC3iEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
March 15, 1979
Pages 343-348
4-NITRO-L-HISTIDINE AS A SUBSTRATE FOR HISTIDINE AMMONIA-LYASE: THE ROLE OF S-HYDROGEN ACIDITY IN THE RATE-LIMITING STEP Claude B. Klee* of Biochemical Pharmacology
Laboratory
Kenneth L. Kirk Laboratory
and Louis A. Cohen of Chemistry
National Institute of Arthritis, Metabolism and Digestive National Institutes of Health, Bethesda, Maryland
February
Received
Diseases, 20014
1,19i'9
Summary: The Km for the interaction of 4-nitro-L-histidine with histidine ammonia-lyase (reduced enzyme, pH 8.0) is comparable to that for L-histidine, while Vmax is l/S that for the natural substrate. With the analog, addition of Cd+2 effects a small decrease in I$a but fails to alter Vmax; the normal deuterium isotope effect for removal of the S-hydrogen (1.5-2.0) is eliminated; and enzyme-catalyzed incorporation of solvent tritium into substrate occurs to a much greater extent than into histidine. Thus, the nitro group increases the acidity of the B-hydrogen and the stability of the conjugate carbanion to such a degree that C-H bond cleavage now precedes rate-limiting C-N bond cleavage.
X=H,F,N02
The detailed respect metal
ion,
certainty
(1).
We had previously
of ammonia
from the
of a small,
initial
of activity,
burst histidine
Present +
is
address:
The nature prosthetic
of histidine
somewhat
group
group--has
atom is at least
demonstration into
of action
of the prosthetic
and a sulfhydryl
g-hydrogen tion
mechanism
to the roles
yet
substrate but
than
argument
isotope
and the observation amino
that
a transition with
reasonable
abstraction
in the rate-limiting
This
reproducible,
faster
(2)
involved (I).+
unidentified),
to be elucidated
demonstrated
partially
ammonia-lyase--with
(yet
that acid
is
of a elimina-
supported
by the
effect,
the absence
solvent
tritium
regeneration
of an
exchange
from urocanate.
*
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 of the interaction between the a-amino group and the enzyme's group is not specified in the structural formulas. 0006-291X/79/050343-06$01.00/0 343
Copyright All rights
@I 1979 by Academic Press, Inc. of reproduction in any form reserved.
BIOCHEMICAL
Vol. 87, No. 1, 1979
4-Fluoro-L-histidine enzyme,
was found
as well
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to be a strong
as a weak substrate.
competitive
The addition
inhibitor
for
the
of Cd+2 has a stimulatory
effect, both for histidine and for its 4-fluoro analog; while the enhancement for fluorohistidine than for histidine, effect of Cd+2 on Km is Ca. 40% greater its effect on V,, is almost 4-fold as great for the analog. This result suggests
that
ammonia
the metal
from
electronic
ion
plays
fluorohistidine,
grounds.
whether
it
have
coupled
to the
a somewhat and fits
The carbanion
the lifetime
more critical
a pattern
formed
which
by B-proton
of an intermediate
substituent
at C-4 both
role
in the removal
can be rationalized abstraction
or a transition
by induction
of on
(II), state,
is
and resonance.
There are indications (3,4) that the electron-releasing resonance effect of fluorine should surpass its inductive effect in such a system, with the result that the fluorine relative have in
atom at C-4 will to that
examined
the latter
group
operate
enhancement MATERIALS
decrease
of histidine.
the acidity
As a logical
test
of the for
this
S-hydrogen interpretation,
the action
of histidine
ammonia-lyase
on 4-nitro-L-histidine;
compound,
the resonance
and inductive
effects
in
the same direction
of B-hydrogen
acidity
and should
produce
atom we
of the nitro
a significant
(5).
AND METHODS:
4-Nitro-L-histidine was prepared as previously &scribed (6). A sample of this compound was stored in 1N NaOD for 2 days at 60'; at this point, the NMR spectrum indicated complete exchange of the B-hydrogens. The solution was acidified to pH 3 and the crystalline, deuterated amino acid was isolated by filtration. Exposure to alkali did not effect racemization at the a-carbon atom, since the deuterated product was totally transformed to nitrourocanate by the enzyme. 4-Nitrourocanic acid was obtained by enzymatic deamination of 4-nitrohistidine. The final reaction mixture was acidified to pH 1 and the crystalline product was separated by filtration, mp 255-263“; --m/e 169. Histidine ammonia-lyase from Pseudomonas ATCC 11299b was prepared as The enzyme was assayed spectrophotometrically at previously described (2,7). as substrate (8). Tbe UV absorption spectra of 277 nm and 25', with histidine 4-nitrohistidine and 4-nitrourocanate show large p&dependent changes between formation of 4-nitrourocanate was pH 7.4 and 9 (Table I); accordingly, followed at different wavelengths depending on pH. The wavelength was chosen to provide maximum UV difference spectra and low absorption of the substrate: 400 nm at pH 8.9-9; 260 nm at pH 8.0; and 370 nm at pH 7.4. UV difference spectra were corrected for the effect of CdC12 on the spectrum of 4-nitrohistidine; the metal ion had no effect on the spectrum of 4-nitrourocanate. The UV spectrum of 4-nitrourocanate is affected by the presence of thiols, and 2-mercaptoethylamine could not be used directly in the assay mixtures. For these studies, the enzyme was reduced and freed of thiol prior to activity Kinetic constants were determined as described (2). measurements (2). RESULTS AND DISCUSSION: With is
histidine
comparable
to that
ammonia-lyase of L-histidine,
at pH 8.0, the Km for and the analog is
344.
4-nitro-L-histidine deaminated at l/8
the
BIOCHEMICAL
Vol. 87, No. 1, 1979
Table
1.
Absorption
Spectral
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Data for 4-Nitrohistidine
and 4-Nitrourocanate.
4-Nitrohistidine Medium
A
pH 9.0)
Tris-IX1
(O.lM,
Tris*HCl
(0.1 M, pH 8.0)
K2HP04 (0.05 M, pH 7.4)
Table
2.
Urocanate
Em x 10
nm
max’
Histidine 4-Fluorohistidine 4-Nitrohistidine
x
Em x 10 -3
nm
IMX’
342
5.58
378
8.68
315
5.55
345
8.40
312
6.11
342
9.18
Formation
from Histidine
and its
Analogs. VlMX
Km bW pH 8.0 pH 8.8
Substrate
4-Nitrourocanate -3
pH 7.4
pH 8.8
pH 8.0
22.80
12.50 0.13 1.60
2.8
1.9
2.0
1.4
0.8
1.0
0.18
4.2
2.1
0.7
1.70
pH
7.4
9.30 0.06
0.94
Reactions were performed in 0.1 M Tris-HCl (pH 8.8 and 8.0) and 0.05 M potassium phosphate (pH 7.41, in the presence of 0.1 mM CdC12. Incubation, at 25', waa started by addition of the substrate; the concentration of reduced enzyme was 10 ug per ml. At high substrate concentrations (0.263 mM), reactions were performed in 2 mm pathlength cuvettes and the enzyme concentration waa raised to 30 ug per ml. V,, is expressed in umoles product/min/mg enzyme.
rate
of the natural
compound in Km with anionic
increasing
Reduction
in both
The value of pK2 for the nitro is Lca 9.5; thus, the marked increase inferior binding (or nonbinding) of the
in catalysis. removal
It were
enzyme,
group
in the presence
found
that
in the rate-limiting with
Km and V,,,
4-nitro-L-histidine are
and a more active per
of added
ion appears therefore,
has been suggested
of the B-proton
results
an oxidized
one sulfhydryl
deamination
max of the metal zyme , addition group is presumed necessary,
ent
II).
anion)
pH may reflect
exists
liberates
for
oxidized
(Table
-f imidazole
species. The lyase
V
substrate
(imidazole
subunit,
to increase the metal step
both
with
the oxidized
V max alone. a metal-ion ion (9).
serves
Km and en-
The sulfhydryl dependent step to facilitate
Significantly
as substrate
the same in the presence
345
form.
improving
Cd+2 (7);
to promote
reduced
(10).
differWith
of EDTA or Cd+2
the
BIOCHEMICAL
Vol. 87, No. 1, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
B 100 l/V
/
I 2
0
1 1 4 6 0 I / [4-NOZ-L-histidine]
Figure 1. Effect of Metal ion reactions were carried out in 0.1 mM CdC12 (0) or 1 mM EDTA A, oxidized enzyme; B, reduced increase in absorbance at 260
(Fig.
lA);
overall
with
rate
rating
the
reduced
of deamination
concentrations
in Km.
Thus,
it
dependence
on metal
the acidity
of the
precedes
ion
for
the rate-limiting
Replacement deuterium
the g-hydrogen
a small
(Fig.
enhancement
lB),
of the
but
this
and is derived,
that
the
proton
4-nitro
abstraction
to such an extent
g-hydrogens
in modest
respectively
6
increase
group or,
at satu-
from a reduction
has either
eliminated
more likely,
that
in the
vanishes
therefore,
The
the
has enhanced
carbanion
formation
now
step.
of the
results
and 2.0,
enzyme Cd+2 effects
appear
S-proton
4
on the deamination of 4-nitro-L-histidine. 0.05 M HEBES-NaOH buffer (pH 7.7) containing (0). The enzyme concentration was 20 ug/ml: enzyme. The velocity is expressed as the nm/min.
of substrate would
2 (mM-‘1
(2).
of histidine
and of 4-fluorohistidine
reductions
in Vmax, with
isotope
This
is
with
our
than
that
fluoro
trend
compound
is
consistent less
acidic
incorporation
of solvent
tritium
into
of 1.5
argument
that
of histidine.
effect is found for the 4-nitro analog (Fig. 2), providing -No isotope evidence for a change in rate-limiting step with this substrate. The enzyme-catalyzed
with
effects
the
further g position
of histidine
and of 4-fluorohistidine demonstrates the reversibility of the With 4-nitrohistidine, the rate of tritium incorporation lyase reaction (2). relative to product formation is 2-3 times as great as for the other subThis result is also consistent with a change in ratestrates (Fig. 3). a significant degree of stability for the carbanion limiting step, and implies
species
in
the nitro
The ability well-established
case.
of the nitro (ll), and is
group to stabilize a carbanion responsible for the acidities
346
by resonance of nitromethane
is
BIOCHEMICAL
Vol. 87, No. 1, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60'/"
I
2
3
I / [4-NOy L-histidine]
4 (mM-’
1
Figure 2. Deuterium isotope effect on the rate of deamination of 4-nitro-Lhistidine. Assays were performed at 25" in 0.05 M HEPES-NaOH buffer (pH 7.6) containing 1 mM EDTA. The reaction was started by addition of oxidized The concentration of substrate was histidine ammonia-lyase (20 ug/ml). measured spectrophotometrically at 312 nm or, after conversion to 4-nitrois expressed as the increase in absorbance urocanate, at 342 nm. The velocity 4-nitro-L-histidine (0). Km = 1.27 + 0.27 mM, Vmax = 0.71 2 at 260 nm/min: 0.09; 6-D2-4-nitro-L-histidine (o), Km = 1.36 + 0.18 mM, Vmax = 0.69 + 0.06.
25
0 TIME
(mm)
Figure 3. Rates of incorporation of solvent tritium into L-histidine, 4-fluoro-L-histidine, and 4-nitro-L-histidine during the histidine ammonialyase reaction. The incubation mixture contained 0.05 M HEPES-NaOH buffer (pH 8), 1 mM mercaptoethylamine, O.lmM CdC12, 4.5 mCi of 3H20, and 32 ug/ml of enzyme. The mixtures were incubated for 10 min at 25' prior to addition of substrate (final concentration 30 mM). At the indicated times, aliquots were removed and incorporation of tritium from water was measured as previously described (2). The formation of 4-nitrourocanate was measured at 400 nm after appropriate dilution in 0.05 M Tris-HCl buffer (pH 9). After repeated lyophilizations of the incubation mixture, 70% of the residual radioactivity was associated with 4-nitrohistidine by thin-layer chromatodemonstrations for the other substrates were described gwhy . Similar previously (2). (pK 10.2) able
and p-nitrotoluene
to the carbanion
6-hydrogens Methods).
is
readily
Indeed,
(pK 28.7)
(5).
of 4-nitrohistidine, achieved
the facility
The same stabilization since
in 1 N aqueous of this
exchange
347
isotope base in
exchange
(see Materials the presence
is
avail-
of the and of the
BIOCHEMICAL
Vol. 87, No. 1, 1979
Table
3.
Effect
of Metal
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Ion on Lyase Activity. Activity
Substrate
Enzyme
Histidine (1.25 u&f) Histidine (1.25 a&I) Nitrohistidine (1.30 r&l) Nitrohistidine (1.30 mN) aExpressed
imidasolate
as umoles
anion for
the
--metal
ioncatalysis,
8-hydrogens,
increases,
effect
(2).to
histidines
is
relationship
of a substituent
in progress,
0.57
0.36
at pH 7.7.
step
effect,
acidity
+ 1 mM EDTA 0.56 0.56 0.29
indicates
isotope
a
4.74 7.81 0.31
an estimated
the kinetic
C-N cleavage throughout
with
deuterium
the rate-limiting
breaking
enzyme
= 1 hr at 60')
in relating
the electronic
oxidized reduced oxidized reduced
product/min/mg
(tl,2
acidity consistent
+O.l ml4 Cd+2
a remarkable
of
pK < 24.
The three criteria and tritium exchange--are
of the
@hydrogen
at C-4 of the ring.
progresses
degree
from
concerted
in histidine
to
As that
acidity
C-H/C-N
bond-
alone.
Examination of other 4-substituted in the hope of demonstrating a linear free
energy
the series.
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11.
Hanson, K. R., and Havir, E. A. (1972) in The Enzymes (Boyer, P. D., ed), 3rd Ed, Vol 7, pp 75-166, Academic Press, New York. Klee, C. B., Kirk, K. L., Cohen, L. A., and McPhie, P. (1975) J. Biol. Chem., 250, 5033-5040. Streitweiser, Jr., A., and Koch, H. F. (1964) J. Am. Chem. Sot., 86, 404-409. 4761-4763. Adolph, H. G., and Kamlet, M. J. (1966) J. Am. Chem. Sot., g, Weiss, C. (1972) Z. Chem., 12, 193-194. Tantz, W., Teitel, S., and Brossi, A. (1973) J. Med. Chem., 16, 705-707. Klee, C. B. (1972) J. Biol. Chem. 247, 1398-1406. Mehler, A. H., and Tabor, H. (1953) J. Biol. Chem., 201, 775-784. A. S., and Abeles, R. H. (1970) Fed. Proc., 29, Givot, I. L., Mildvan, 1590. Klee, C. B., Kirk, K. L., McPhie, P., and Cohen, L. A. (1974) Fed. Proc., 22, 1318. Cram, D. J. (1965) Fundamentals of Carbanion Chemistry, pp 52-55, Academic Press, New York.
348
8lOC3iEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
March 15, 1979
Pages 343-348
4-NITRO-L-HISTIDINE AS A SUBSTRATE FOR HISTIDINE AMMONIA-LYASE: THE ROLE OF S-HYDROGEN ACIDITY IN THE RATE-LIMITING STEP Claude B. Klee* of Biochemical Pharmacology
Laboratory
Kenneth L. Kirk Laboratory
and Louis A. Cohen of Chemistry
National Institute of Arthritis, Metabolism and Digestive National Institutes of Health, Bethesda, Maryland
February
Received
Diseases, 20014
1,19i'9
Summary: The Km for the interaction of 4-nitro-L-histidine with histidine ammonia-lyase (reduced enzyme, pH 8.0) is comparable to that for L-histidine, while Vmax is l/S that for the natural substrate. With the analog, addition of Cd+2 effects a small decrease in I$a but fails to alter Vmax; the normal deuterium isotope effect for removal of the S-hydrogen (1.5-2.0) is eliminated; and enzyme-catalyzed incorporation of solvent tritium into substrate occurs to a much greater extent than into histidine. Thus, the nitro group increases the acidity of the B-hydrogen and the stability of the conjugate carbanion to such a degree that C-H bond cleavage now precedes rate-limiting C-N bond cleavage.
X=H,F,N02
The detailed respect metal
ion,
certainty
(1).
We had previously
of ammonia
from the
of a small,
initial
of activity,
burst histidine
Present +
is
address:
The nature prosthetic
of histidine
somewhat
group
group--has
atom is at least
demonstration into
of action
of the prosthetic
and a sulfhydryl
g-hydrogen tion
mechanism
to the roles
yet
substrate but
than
argument
isotope
and the observation amino
that
a transition with
reasonable
abstraction
in the rate-limiting
This
reproducible,
faster
(2)
involved (I).+
unidentified),
to be elucidated
demonstrated
partially
ammonia-lyase--with
(yet
that acid
is
of a elimina-
supported
by the
effect,
the absence
solvent
tritium
regeneration
of an
exchange
from urocanate.
*
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 of the interaction between the a-amino group and the enzyme's group is not specified in the structural formulas. 0006-291X/79/050343-06$01.00/0 343
Copyright All rights
@I 1979 by Academic Press, Inc. of reproduction in any form reserved.
BIOCHEMICAL
Vol. 87, No. 1, 1979
4-Fluoro-L-histidine enzyme,
was found
as well
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to be a strong
as a weak substrate.
competitive
The addition
inhibitor
for
the
of Cd+2 has a stimulatory
effect, both for histidine and for its 4-fluoro analog; while the enhancement for fluorohistidine than for histidine, effect of Cd+2 on Km is Ca. 40% greater its effect on V,, is almost 4-fold as great for the analog. This result suggests
that
ammonia
the metal
from
electronic
ion
plays
fluorohistidine,
grounds.
whether
it
have
coupled
to the
a somewhat and fits
The carbanion
the lifetime
more critical
a pattern
formed
which
by B-proton
of an intermediate
substituent
at C-4 both
role
in the removal
can be rationalized abstraction
or a transition
by induction
of on
(II), state,
is
and resonance.
There are indications (3,4) that the electron-releasing resonance effect of fluorine should surpass its inductive effect in such a system, with the result that the fluorine relative have in
atom at C-4 will to that
examined
the latter
group
operate
enhancement MATERIALS
decrease
of histidine.
the acidity
As a logical
test
of the for
this
S-hydrogen interpretation,
the action
of histidine
ammonia-lyase
on 4-nitro-L-histidine;
compound,
the resonance
and inductive
effects
in
the same direction
of B-hydrogen
acidity
and should
produce
atom we
of the nitro
a significant
(5).
AND METHODS:
4-Nitro-L-histidine was prepared as previously &scribed (6). A sample of this compound was stored in 1N NaOD for 2 days at 60'; at this point, the NMR spectrum indicated complete exchange of the B-hydrogens. The solution was acidified to pH 3 and the crystalline, deuterated amino acid was isolated by filtration. Exposure to alkali did not effect racemization at the a-carbon atom, since the deuterated product was totally transformed to nitrourocanate by the enzyme. 4-Nitrourocanic acid was obtained by enzymatic deamination of 4-nitrohistidine. The final reaction mixture was acidified to pH 1 and the crystalline product was separated by filtration, mp 255-263“; --m/e 169. Histidine ammonia-lyase from Pseudomonas ATCC 11299b was prepared as The enzyme was assayed spectrophotometrically at previously described (2,7). as substrate (8). Tbe UV absorption spectra of 277 nm and 25', with histidine 4-nitrohistidine and 4-nitrourocanate show large p&dependent changes between formation of 4-nitrourocanate was pH 7.4 and 9 (Table I); accordingly, followed at different wavelengths depending on pH. The wavelength was chosen to provide maximum UV difference spectra and low absorption of the substrate: 400 nm at pH 8.9-9; 260 nm at pH 8.0; and 370 nm at pH 7.4. UV difference spectra were corrected for the effect of CdC12 on the spectrum of 4-nitrohistidine; the metal ion had no effect on the spectrum of 4-nitrourocanate. The UV spectrum of 4-nitrourocanate is affected by the presence of thiols, and 2-mercaptoethylamine could not be used directly in the assay mixtures. For these studies, the enzyme was reduced and freed of thiol prior to activity Kinetic constants were determined as described (2). measurements (2). RESULTS AND DISCUSSION: With is
histidine
comparable
to that
ammonia-lyase of L-histidine,
at pH 8.0, the Km for and the analog is
344.
4-nitro-L-histidine deaminated at l/8
the
BIOCHEMICAL
Vol. 87, No. 1, 1979
Table
1.
Absorption
Spectral
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Data for 4-Nitrohistidine
and 4-Nitrourocanate.
4-Nitrohistidine Medium
A
pH 9.0)
Tris-IX1
(O.lM,
Tris*HCl
(0.1 M, pH 8.0)
K2HP04 (0.05 M, pH 7.4)
Table
2.
Urocanate
Em x 10
nm
max’
Histidine 4-Fluorohistidine 4-Nitrohistidine
x
Em x 10 -3
nm
IMX’
342
5.58
378
8.68
315
5.55
345
8.40
312
6.11
342
9.18
Formation
from Histidine
and its
Analogs. VlMX
Km bW pH 8.0 pH 8.8
Substrate
4-Nitrourocanate -3
pH 7.4
pH 8.8
pH 8.0
22.80
12.50 0.13 1.60
2.8
1.9
2.0
1.4
0.8
1.0
0.18
4.2
2.1
0.7
1.70
pH
7.4
9.30 0.06
0.94
Reactions were performed in 0.1 M Tris-HCl (pH 8.8 and 8.0) and 0.05 M potassium phosphate (pH 7.41, in the presence of 0.1 mM CdC12. Incubation, at 25', waa started by addition of the substrate; the concentration of reduced enzyme was 10 ug per ml. At high substrate concentrations (0.263 mM), reactions were performed in 2 mm pathlength cuvettes and the enzyme concentration waa raised to 30 ug per ml. V,, is expressed in umoles product/min/mg enzyme.
rate
of the natural
compound in Km with anionic
increasing
Reduction
in both
The value of pK2 for the nitro is Lca 9.5; thus, the marked increase inferior binding (or nonbinding) of the
in catalysis. removal
It were
enzyme,
group
in the presence
found
that
in the rate-limiting with
Km and V,,,
4-nitro-L-histidine are
and a more active per
of added
ion appears therefore,
has been suggested
of the B-proton
results
an oxidized
one sulfhydryl
deamination
max of the metal zyme , addition group is presumed necessary,
ent
II).
anion)
pH may reflect
exists
liberates
for
oxidized
(Table
-f imidazole
species. The lyase
V
substrate
(imidazole
subunit,
to increase the metal step
both
with
the oxidized
V max alone. a metal-ion ion (9).
serves
Km and en-
The sulfhydryl dependent step to facilitate
Significantly
as substrate
the same in the presence
345
form.
improving
Cd+2 (7);
to promote
reduced
(10).
differWith
of EDTA or Cd+2
the
BIOCHEMICAL
Vol. 87, No. 1, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
B 100 l/V
/
I 2
0
1 1 4 6 0 I / [4-NOZ-L-histidine]
Figure 1. Effect of Metal ion reactions were carried out in 0.1 mM CdC12 (0) or 1 mM EDTA A, oxidized enzyme; B, reduced increase in absorbance at 260
(Fig.
lA);
overall
with
rate
rating
the
reduced
of deamination
concentrations
in Km.
Thus,
it
dependence
on metal
the acidity
of the
precedes
ion
for
the rate-limiting
Replacement deuterium
the g-hydrogen
a small
(Fig.
enhancement
lB),
of the
but
this
and is derived,
that
the
proton
4-nitro
abstraction
to such an extent
g-hydrogens
in modest
respectively
6
increase
group or,
at satu-
from a reduction
has either
eliminated
more likely,
that
in the
vanishes
therefore,
The
the
has enhanced
carbanion
formation
now
step.
of the
results
and 2.0,
enzyme Cd+2 effects
appear
S-proton
4
on the deamination of 4-nitro-L-histidine. 0.05 M HEBES-NaOH buffer (pH 7.7) containing (0). The enzyme concentration was 20 ug/ml: enzyme. The velocity is expressed as the nm/min.
of substrate would
2 (mM-‘1
(2).
of histidine
and of 4-fluorohistidine
reductions
in Vmax, with
isotope
This
is
with
our
than
that
fluoro
trend
compound
is
consistent less
acidic
incorporation
of solvent
tritium
into
of 1.5
argument
that
of histidine.
effect is found for the 4-nitro analog (Fig. 2), providing -No isotope evidence for a change in rate-limiting step with this substrate. The enzyme-catalyzed
with
effects
the
further g position
of histidine
and of 4-fluorohistidine demonstrates the reversibility of the With 4-nitrohistidine, the rate of tritium incorporation lyase reaction (2). relative to product formation is 2-3 times as great as for the other subThis result is also consistent with a change in ratestrates (Fig. 3). a significant degree of stability for the carbanion limiting step, and implies
species
in
the nitro
The ability well-established
case.
of the nitro (ll), and is
group to stabilize a carbanion responsible for the acidities
346
by resonance of nitromethane
is
BIOCHEMICAL
Vol. 87, No. 1, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60'/"
I
2
3
I / [4-NOy L-histidine]
4 (mM-’
1
Figure 2. Deuterium isotope effect on the rate of deamination of 4-nitro-Lhistidine. Assays were performed at 25" in 0.05 M HEPES-NaOH buffer (pH 7.6) containing 1 mM EDTA. The reaction was started by addition of oxidized The concentration of substrate was histidine ammonia-lyase (20 ug/ml). measured spectrophotometrically at 312 nm or, after conversion to 4-nitrois expressed as the increase in absorbance urocanate, at 342 nm. The velocity 4-nitro-L-histidine (0). Km = 1.27 + 0.27 mM, Vmax = 0.71 2 at 260 nm/min: 0.09; 6-D2-4-nitro-L-histidine (o), Km = 1.36 + 0.18 mM, Vmax = 0.69 + 0.06.
25
0 TIME
(mm)
Figure 3. Rates of incorporation of solvent tritium into L-histidine, 4-fluoro-L-histidine, and 4-nitro-L-histidine during the histidine ammonialyase reaction. The incubation mixture contained 0.05 M HEPES-NaOH buffer (pH 8), 1 mM mercaptoethylamine, O.lmM CdC12, 4.5 mCi of 3H20, and 32 ug/ml of enzyme. The mixtures were incubated for 10 min at 25' prior to addition of substrate (final concentration 30 mM). At the indicated times, aliquots were removed and incorporation of tritium from water was measured as previously described (2). The formation of 4-nitrourocanate was measured at 400 nm after appropriate dilution in 0.05 M Tris-HCl buffer (pH 9). After repeated lyophilizations of the incubation mixture, 70% of the residual radioactivity was associated with 4-nitrohistidine by thin-layer chromatodemonstrations for the other substrates were described gwhy . Similar previously (2). (pK 10.2) able
and p-nitrotoluene
to the carbanion
6-hydrogens Methods).
is
readily
Indeed,
(pK 28.7)
(5).
of 4-nitrohistidine, achieved
the facility
The same stabilization since
in 1 N aqueous of this
exchange
347
isotope base in
exchange
(see Materials the presence
is
avail-
of the and of the
BIOCHEMICAL
Vol. 87, No. 1, 1979
Table
3.
Effect
of Metal
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Ion on Lyase Activity. Activity
Substrate
Enzyme
Histidine (1.25 u&f) Histidine (1.25 a&I) Nitrohistidine (1.30 r&l) Nitrohistidine (1.30 mN) aExpressed
imidasolate
as umoles
anion for
the
--metal
ioncatalysis,
8-hydrogens,
increases,
effect
(2).to
histidines
is
relationship
of a substituent
in progress,
0.57
0.36
at pH 7.7.
step
effect,
acidity
+ 1 mM EDTA 0.56 0.56 0.29
indicates
isotope
a
4.74 7.81 0.31
an estimated
the kinetic
C-N cleavage throughout
with
deuterium
the rate-limiting
breaking
enzyme
= 1 hr at 60')
in relating
the electronic
oxidized reduced oxidized reduced
product/min/mg
(tl,2
acidity consistent
+O.l ml4 Cd+2
a remarkable
of
pK < 24.
The three criteria and tritium exchange--are
of the
@hydrogen
at C-4 of the ring.
progresses
degree
from
concerted
in histidine
to
As that
acidity
C-H/C-N
bond-
alone.
Examination of other 4-substituted in the hope of demonstrating a linear free
energy
the series.
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11.
Hanson, K. R., and Havir, E. A. (1972) in The Enzymes (Boyer, P. D., ed), 3rd Ed, Vol 7, pp 75-166, Academic Press, New York. Klee, C. B., Kirk, K. L., Cohen, L. A., and McPhie, P. (1975) J. Biol. Chem., 250, 5033-5040. Streitweiser, Jr., A., and Koch, H. F. (1964) J. Am. Chem. Sot., 86, 404-409. 4761-4763. Adolph, H. G., and Kamlet, M. J. (1966) J. Am. Chem. Sot., g, Weiss, C. (1972) Z. Chem., 12, 193-194. Tantz, W., Teitel, S., and Brossi, A. (1973) J. Med. Chem., 16, 705-707. Klee, C. B. (1972) J. Biol. Chem. 247, 1398-1406. Mehler, A. H., and Tabor, H. (1953) J. Biol. Chem., 201, 775-784. A. S., and Abeles, R. H. (1970) Fed. Proc., 29, Givot, I. L., Mildvan, 1590. Klee, C. B., Kirk, K. L., McPhie, P., and Cohen, L. A. (1974) Fed. Proc., 22, 1318. Cram, D. J. (1965) Fundamentals of Carbanion Chemistry, pp 52-55, Academic Press, New York.
348
