Vol. 68, No. 3, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FUNCTIONAL ARGININE RESIDUES INVOLVED IN COENZYME BINDING BY DIHYDROFOLATE REDUCTASE
Gordon
A. Vehari
and James H. Freisheim*
Department of Biological Chemistry, College of Medicine University of Cincinnati, Cincinnati, Ohio 45267 Received
December
8,1975 SUMMARY
Reaction of dihydrofolate reductase from amethopterin-resistant Lactobacillus casei with phenylglyoxal results in a complete loss of enzyme activity, This inactivation is concomitant with the modification of five of a total of eight arginine residues per mole of enzyme. In the presence of the reduced coenzyme, NADPH, two of the five reactive arginines are protected from chemical modification with complete retention of enzyme activity. The results suggest the involvement of essential arginine residues at or near the coenzyme binding site and thus at or near the active center of the enzyme. INTRODUCTION Arginine of pyridine found
residues
modification
ase by butanedione of the
(2) have phosphate
to be involved
nucleotide-dependent
that
studies
appear
shown
resulted
that
bridge
implicated
(4)
The reduced
tetrahydrofolate,
M4 lactate
for
ca.
moves
binding
as well
as in malate
coenzyme,
NADPH, is
the reduction the coenzyme
X-ray
carrier
*Present address, Department University of Washington, Seattle,
arginine
dehydrogenases
of L-7,8
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
dehydrogen-
the
residues (3),
pyro-
have been
glutamate
(5). reductase
to l,L-5,6,7,8-
of Biochemistry, School Washington 98195.
937
with
in a variety
*Research Career Development Awardee of the U.S. (CA-70449); to whom inquiries should be addressed.
(1)
by Adams --et al.
by dihydrofolate
dihydrofolate involved
a number
crystallographic
complex
dehydrogenases
required
in
H4 lactate
13 A to interact
In addition,
in alcohol
binding
Yang and Schwert
of bovine
dehydrogenase-NAD
arginine-101
in coenzyme
residues
(l-5).
in enzyme inactivation.
of the coenzyme.
dehydrogenases
(E.C.1.5.1.3)
dehydrogenases
of arginine
dogfish
in coenzyme
of one-carbon of Medicine,
Public
Health
Service
Vol. 68, No. 3, 1976
transfer role
BIOCHEMICAL
reactions.
In the
of arginine
evaluated
residues
AND BIOPHYSICAL
present
communication
in coenzyme
by means of chemical
binding
the possible
functional
by dihydrofolate
modification
MATERIALS
RESEARCH COMMUNICATIONS
with
reductase
is
phenylglyoxal.
AND METHODS
Dihydrofolate reductase from an amethopterin-resistant strain of Lactobacillus casei was isolated and purified by a modification (6) of the procedure of Gunderson, Electrophoretic form I, which does not --et al. (7). contain bound NADPH, was employed in these studies. Enzyme assays were performed spectrophotometrically at 25' by following the decrease in absorbance at 340 nm using a Gilford Model 240 spectrophotometer and a Sargent Model SRLG recorder. The assay mixture (1.2 ml) contained 60 mmoles KP04 buffer, pH 7.0, 50 nmoles of NADPH (P-L Biochemicals), 40 nmoles of dihydrofolate and Dihydrofolate was prepared by the reduction of folic enzyme (ca. 1 x 10W7g). acid (Cycle chemicals) with sodium dithionite according to the method of Blakley (8). Enzyme modifications with phenylglyoxal (K and K Labs) were done at 20' in 0.1 M KP04, pH 7.8. Amino acid analyses were performed in a Durrum D-500 amino acid analyzer following hydrolysis --in vacua in 6 1 HCl for 20 hr at 110' according to the general procedures of Moore and Stein (9).
RESULTS AND DISCUSSION Solutions 200-fold
molar
identical
excess
conditions
glyoxal
1.
ratio
without
reductase
where
Aliquots intervals
at each
interval
hydrolysis sample.
remaining
the time
is
precipitated
As indicated corresponds
per mole enzymes
Many of the highly
in Figure
of protein.. indicate polar
2, the
to a loss Comparison that
guanidino
no amino groups 938
as the log
mixture
were
inactivation.
of the
of the analyses
of arginine
after
than
at
acid
was performed
inactivation
of a total
other
removed The reaction
analysis
complete
acid
shown in
phenylglyoxal-modified
acid
of five
under
of phenyl-
is
of 6 E HCl and,
amino
a
control.
of the
protein,
with
incubated
The effect
reaction
by the addition
were
expressed
of the
course
incubated
reductase
of an unreacted
was terminated
reductase
inactivated
of reagent.
the enzyme activity
the activity
were
samples
of dihydrofolate
activity
during
of the
residues
Control
of the enzyme-phenylglyoxal
various
folate
V is
and Vo is
(5 x 10e5@
the addition
activity
The fractional
V/Vo,
reductase
of phenylglyoxal.
on the enzymatic
Figure
each
of dihydrofolate
of dihydro-
of eight for
arginine
the native
arginine
residues
on
would,
is
and modified. presum-
Vol. 68, No. 3, 1976
BIOCHEMICAL
1 200
0
1
1
400 min.
Time,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
600 Arg
Residues
Modified/Mole
of dihydrofolate reductase by phenylglyoxal. Enzyme FIGURE 1. Inactivation (2 x 10-5M> in O.lM KP04, p H 7.8, was incubated with 4 x lo-3 phenylglyoxal at 20'. Aliquots were removed at the indicated times for assay. FIGURE 2. Decrease of dihydrofolate arginine modification. Enzyme (5 x excess of phenylglyoxal as described mixture were treated as described in to determine the residues of arginine
ably,
be exposed
half
of the total
on the surtace arginines
In an attempt residues, nucleotide
prior
NADPH prior against are
enzyme inactivation.
modified
removal
inactivated residues
without
in
loss
in
the presence
to coenzyme
binding
results
activity
and reduced
and are,
reduced
in complete three
coenzyme,
therefore,
pyridine
arginine
Of the five
that
these
at or near
are
with
protection
the reductase
two such residues
of NADPH, suggesting
arginine
as shown in Figure
of phenylglyoxal. reductase,
more than
of the reductase
approximately
in enzymatic
the presence
with
Incubation
phenylglyoxal
of the reagent
in dihydrofolate
from non-essential
was pre-treated
However,
work
by phenylglyoxal.
modification.
with
In the present
essential
reductase
to treatment
Following
ible
are modified
to chemical
activity as a function of reacted with a 200-fold molar 1. Aliquots of the inhibition and amino acid analyses performed
of the protein.
to distinguish
dihydrofolate
reductase 10e5M) was in Figure the text present.
residues 3. is
reactive
arginine
rendered
inaccess-
residues the active
are essential center
of the
enzyme.
group
Recently,
Feeney --et al.
(10)
of either
NADPH or NADP+ is
have
demonstrated
in the dianionic
939
that state
the
2'-phosphate
in binding
complexes
Vol. 68, No. 3,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.0
0.2 0.8
\. 2
Arg
4
Residues
6
8
Modified/
Mole
FIGURE 3. Effect of phenylglyoxal on the activity of dihydrofolate reductase in the presence and absence of NADPH. Enzyme (5 x 10B5M) was incubated for five minutes with 5 x 10% NADPH in 0.e KP04, pH 7.8,prior to the addition of a ZOO-fold molar excess of phenylglyoxal to enzyme. Following reaction for two hours at 20' the reaction mixture was passed through a 2.5 x 45 cm Sephadex G-25 column equilibrated with pH 7.8 phosphate buffer to remove the reagent and NADPH. The enzyme was again treated with phenylglyoxal as described above; enzyme activity and arginine contents were determined as described in Figures 1 and 2, respectively.
with
&. casei
chemical
dihydrofolate
shift
reductase,
and position
enzyme complex
is
that
the pK of the
that
found
for
resonance
of pH in the
of 4.5
2'-phosphate
drastic
alteration
support
the possibility
group
of the in
free
groups moiety
strong
salt
a dianionic
contribution electrostatic
and arginine dihydrofolate in
with
the binding Studies
residues reductase.
in
2'-phosphate
between
a role
now in progress
group
to determine
940
been shown
the
location
from
this
moiety
would
form
group binding
In
of NADPH by
to be important indicated. of these
a
make a
binding.
pyrophosphace
as was previously
units
therefore,
nucleotide
have
indicating
positive
residues
of dinucleotide
the
dinucleotide
strongly
between
and,
The
Such a
The permanent
in pyridine
Such interactions
three
would
interaction
of the energy
of NAD+ to dehydrogenases are
group
phosphoryl
interactions
to 7.5,
in solution.
of one or more arginine
terms
may play
alone
data .
in either
by at least
on the protein.
of the guanidino
addition,
differs
coenzymes
the pK of the
charge
significant
range
of an electrostatic
and one or more cationic
bridge
on 31P NMR spectral
of the 2'-phosphate
independent
either
based
arginine
Vol. 68, No. 3, 1976
residues
in
localization
BIOCHEMICAL
the amino
acid
sequence
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of dihydrofolate
of one or more arginine
in the NADPH binding
domain
structural
phosphorylated
basis
for
may well
reductase.
residues
near
provide,
at least
dinucleotide
the coensyme in part,
binding
The unique 2'-phosphate the
by dihydrofolate
reductase
ACKNOWLEDGEMENTS This Institute,
research United
was supported States
Public
by Grant Health
CA-11666
from
the National
Cancer
Service.
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10.
Yang, P. C., and Schwert, G. W. (1972) Biochemistry, 11, 2218. Adams, M. J., Buehner, M., Chandrasekhar, K., Ford, G. C., Hackert, M. L., Liljas, A., Rossmann, M. G., Smiley, I. E., Allison, W. S., Everse, J., Kaplan, N. 0. and Taylor, S. S. (1973) Proc. Nat. Acad. Sci. U.S.A., 70, 1968. J. F. and Vallee, B. L. (1974) Biochemistry, 13, 4361. Lang, L. G., Riordan, Blumenthal, K. M. and Smith, E. L. (1975) J. Biol. Chem., 250, 6555. Bleile, D. M., Foster, M., Brady, J. W. and Harrison, J. H. (1975) J. Biol. Chem., 250, 6222. Liu, J.-K. and Dunlap, R. B. (1974) Biochemistry, 13, 1807. Gundersen, L. E., Dunlap, R. B., Harding, N. G. L., Freisheim, J. H., Otting, F. and Huennekens, F. M. (1972) Biochemistry, 11, 1018. Blakley, R. L. (1960) Nature, 188, 231. Moore, S. and Stein, W. H. (1963) Methods Enzymol., 6, 819. Feeney, J., Birdsall, B., Roberts, G. C. K. and Burgen, A. S. V. (1975) Nature, 257, 564.
941
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FUNCTIONAL ARGININE RESIDUES INVOLVED IN COENZYME BINDING BY DIHYDROFOLATE REDUCTASE
Gordon
A. Vehari
and James H. Freisheim*
Department of Biological Chemistry, College of Medicine University of Cincinnati, Cincinnati, Ohio 45267 Received
December
8,1975 SUMMARY
Reaction of dihydrofolate reductase from amethopterin-resistant Lactobacillus casei with phenylglyoxal results in a complete loss of enzyme activity, This inactivation is concomitant with the modification of five of a total of eight arginine residues per mole of enzyme. In the presence of the reduced coenzyme, NADPH, two of the five reactive arginines are protected from chemical modification with complete retention of enzyme activity. The results suggest the involvement of essential arginine residues at or near the coenzyme binding site and thus at or near the active center of the enzyme. INTRODUCTION Arginine of pyridine found
residues
modification
ase by butanedione of the
(2) have phosphate
to be involved
nucleotide-dependent
that
studies
appear
shown
resulted
that
bridge
implicated
(4)
The reduced
tetrahydrofolate,
M4 lactate
for
ca.
moves
binding
as well
as in malate
coenzyme,
NADPH, is
the reduction the coenzyme
X-ray
carrier
*Present address, Department University of Washington, Seattle,
arginine
dehydrogenases
of L-7,8
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
dehydrogen-
the
residues (3),
pyro-
have been
glutamate
(5). reductase
to l,L-5,6,7,8-
of Biochemistry, School Washington 98195.
937
with
in a variety
*Research Career Development Awardee of the U.S. (CA-70449); to whom inquiries should be addressed.
(1)
by Adams --et al.
by dihydrofolate
dihydrofolate involved
a number
crystallographic
complex
dehydrogenases
required
in
H4 lactate
13 A to interact
In addition,
in alcohol
binding
Yang and Schwert
of bovine
dehydrogenase-NAD
arginine-101
in coenzyme
residues
(l-5).
in enzyme inactivation.
of the coenzyme.
dehydrogenases
(E.C.1.5.1.3)
dehydrogenases
of arginine
dogfish
in coenzyme
of one-carbon of Medicine,
Public
Health
Service
Vol. 68, No. 3, 1976
transfer role
BIOCHEMICAL
reactions.
In the
of arginine
evaluated
residues
AND BIOPHYSICAL
present
communication
in coenzyme
by means of chemical
binding
the possible
functional
by dihydrofolate
modification
MATERIALS
RESEARCH COMMUNICATIONS
with
reductase
is
phenylglyoxal.
AND METHODS
Dihydrofolate reductase from an amethopterin-resistant strain of Lactobacillus casei was isolated and purified by a modification (6) of the procedure of Gunderson, Electrophoretic form I, which does not --et al. (7). contain bound NADPH, was employed in these studies. Enzyme assays were performed spectrophotometrically at 25' by following the decrease in absorbance at 340 nm using a Gilford Model 240 spectrophotometer and a Sargent Model SRLG recorder. The assay mixture (1.2 ml) contained 60 mmoles KP04 buffer, pH 7.0, 50 nmoles of NADPH (P-L Biochemicals), 40 nmoles of dihydrofolate and Dihydrofolate was prepared by the reduction of folic enzyme (ca. 1 x 10W7g). acid (Cycle chemicals) with sodium dithionite according to the method of Blakley (8). Enzyme modifications with phenylglyoxal (K and K Labs) were done at 20' in 0.1 M KP04, pH 7.8. Amino acid analyses were performed in a Durrum D-500 amino acid analyzer following hydrolysis --in vacua in 6 1 HCl for 20 hr at 110' according to the general procedures of Moore and Stein (9).
RESULTS AND DISCUSSION Solutions 200-fold
molar
identical
excess
conditions
glyoxal
1.
ratio
without
reductase
where
Aliquots intervals
at each
interval
hydrolysis sample.
remaining
the time
is
precipitated
As indicated corresponds
per mole enzymes
Many of the highly
in Figure
of protein.. indicate polar
2, the
to a loss Comparison that
guanidino
no amino groups 938
as the log
mixture
were
inactivation.
of the
of the analyses
of arginine
after
than
at
acid
was performed
inactivation
of a total
other
removed The reaction
analysis
complete
acid
shown in
phenylglyoxal-modified
acid
of five
under
of phenyl-
is
of 6 E HCl and,
amino
a
control.
of the
protein,
with
incubated
The effect
reaction
by the addition
were
expressed
of the
course
incubated
reductase
of an unreacted
was terminated
reductase
inactivated
of reagent.
the enzyme activity
the activity
were
samples
of dihydrofolate
activity
during
of the
residues
Control
of the enzyme-phenylglyoxal
various
folate
V is
and Vo is
(5 x 10e5@
the addition
activity
The fractional
V/Vo,
reductase
of phenylglyoxal.
on the enzymatic
Figure
each
of dihydrofolate
of dihydro-
of eight for
arginine
the native
arginine
residues
on
would,
is
and modified. presum-
Vol. 68, No. 3, 1976
BIOCHEMICAL
1 200
0
1
1
400 min.
Time,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
600 Arg
Residues
Modified/Mole
of dihydrofolate reductase by phenylglyoxal. Enzyme FIGURE 1. Inactivation (2 x 10-5M> in O.lM KP04, p H 7.8, was incubated with 4 x lo-3 phenylglyoxal at 20'. Aliquots were removed at the indicated times for assay. FIGURE 2. Decrease of dihydrofolate arginine modification. Enzyme (5 x excess of phenylglyoxal as described mixture were treated as described in to determine the residues of arginine
ably,
be exposed
half
of the total
on the surtace arginines
In an attempt residues, nucleotide
prior
NADPH prior against are
enzyme inactivation.
modified
removal
inactivated residues
without
in
loss
in
the presence
to coenzyme
binding
results
activity
and reduced
and are,
reduced
in complete three
coenzyme,
therefore,
pyridine
arginine
Of the five
that
these
at or near
are
with
protection
the reductase
two such residues
of NADPH, suggesting
arginine
as shown in Figure
of phenylglyoxal. reductase,
more than
of the reductase
approximately
in enzymatic
the presence
with
Incubation
phenylglyoxal
of the reagent
in dihydrofolate
from non-essential
was pre-treated
However,
work
by phenylglyoxal.
modification.
with
In the present
essential
reductase
to treatment
Following
ible
are modified
to chemical
activity as a function of reacted with a 200-fold molar 1. Aliquots of the inhibition and amino acid analyses performed
of the protein.
to distinguish
dihydrofolate
reductase 10e5M) was in Figure the text present.
residues 3. is
reactive
arginine
rendered
inaccess-
residues the active
are essential center
of the
enzyme.
group
Recently,
Feeney --et al.
(10)
of either
NADPH or NADP+ is
have
demonstrated
in the dianionic
939
that state
the
2'-phosphate
in binding
complexes
Vol. 68, No. 3,1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.0
0.2 0.8
\. 2
Arg
4
Residues
6
8
Modified/
Mole
FIGURE 3. Effect of phenylglyoxal on the activity of dihydrofolate reductase in the presence and absence of NADPH. Enzyme (5 x 10B5M) was incubated for five minutes with 5 x 10% NADPH in 0.e KP04, pH 7.8,prior to the addition of a ZOO-fold molar excess of phenylglyoxal to enzyme. Following reaction for two hours at 20' the reaction mixture was passed through a 2.5 x 45 cm Sephadex G-25 column equilibrated with pH 7.8 phosphate buffer to remove the reagent and NADPH. The enzyme was again treated with phenylglyoxal as described above; enzyme activity and arginine contents were determined as described in Figures 1 and 2, respectively.
with
&. casei
chemical
dihydrofolate
shift
reductase,
and position
enzyme complex
is
that
the pK of the
that
found
for
resonance
of pH in the
of 4.5
2'-phosphate
drastic
alteration
support
the possibility
group
of the in
free
groups moiety
strong
salt
a dianionic
contribution electrostatic
and arginine dihydrofolate in
with
the binding Studies
residues reductase.
in
2'-phosphate
between
a role
now in progress
group
to determine
940
been shown
the
location
from
this
moiety
would
form
group binding
In
of NADPH by
to be important indicated. of these
a
make a
binding.
pyrophosphace
as was previously
units
therefore,
nucleotide
have
indicating
positive
residues
of dinucleotide
the
dinucleotide
strongly
between
and,
The
Such a
The permanent
in pyridine
Such interactions
three
would
interaction
of the energy
of NAD+ to dehydrogenases are
group
phosphoryl
interactions
to 7.5,
in solution.
of one or more arginine
terms
may play
alone
data .
in either
by at least
on the protein.
of the guanidino
addition,
differs
coenzymes
the pK of the
charge
significant
range
of an electrostatic
and one or more cationic
bridge
on 31P NMR spectral
of the 2'-phosphate
independent
either
based
arginine
Vol. 68, No. 3, 1976
residues
in
localization
BIOCHEMICAL
the amino
acid
sequence
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of dihydrofolate
of one or more arginine
in the NADPH binding
domain
structural
phosphorylated
basis
for
may well
reductase.
residues
near
provide,
at least
dinucleotide
the coensyme in part,
binding
The unique 2'-phosphate the
by dihydrofolate
reductase
ACKNOWLEDGEMENTS This Institute,
research United
was supported States
Public
by Grant Health
CA-11666
from
the National
Cancer
Service.
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10.
Yang, P. C., and Schwert, G. W. (1972) Biochemistry, 11, 2218. Adams, M. J., Buehner, M., Chandrasekhar, K., Ford, G. C., Hackert, M. L., Liljas, A., Rossmann, M. G., Smiley, I. E., Allison, W. S., Everse, J., Kaplan, N. 0. and Taylor, S. S. (1973) Proc. Nat. Acad. Sci. U.S.A., 70, 1968. J. F. and Vallee, B. L. (1974) Biochemistry, 13, 4361. Lang, L. G., Riordan, Blumenthal, K. M. and Smith, E. L. (1975) J. Biol. Chem., 250, 6555. Bleile, D. M., Foster, M., Brady, J. W. and Harrison, J. H. (1975) J. Biol. Chem., 250, 6222. Liu, J.-K. and Dunlap, R. B. (1974) Biochemistry, 13, 1807. Gundersen, L. E., Dunlap, R. B., Harding, N. G. L., Freisheim, J. H., Otting, F. and Huennekens, F. M. (1972) Biochemistry, 11, 1018. Blakley, R. L. (1960) Nature, 188, 231. Moore, S. and Stein, W. H. (1963) Methods Enzymol., 6, 819. Feeney, J., Birdsall, B., Roberts, G. C. K. and Burgen, A. S. V. (1975) Nature, 257, 564.
941
