BIOCHEMICAL
Vol. 87, No. 1, 1979 March.15,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
1979
ESSENTIAL
CARBOXY
Alfred Department Received
L.
George,
of Chemistry,
January
L RESIDUES 2
Jr.
IN YEAST
and C.
College
L.
ENOLASE’
Borders,
of Wooster,
59-65
3
Jr.
Wooster,
Ohio
44691
19,1979
Yeast enolase (EC 4.2.1.11) is rapidly inactivated at pH 6.1 by three different water-soluble carbodiimides -- 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, and l-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide. Inactivation is most likely due to the modification of essential carboxyl residues at the enzyme active site. Chemical essential
for
modification
studies
have
the catalytic
activity
of yeast
sential
arginyl
residue
anionic
phosphate
has been
shown
histidine
is less
related
with
under
It has been muscle
However,
selective study
well
essential
defined.
in the catalytic
this
enolase for suggests
binding that
by reaction
reagent
fails
several
the modification that
yeast
The
enzyme
is likely
in neither
(7,8) are
residues
in binding
enolase played
has been
and cysteinyl
es-
the
In addition, but the role
by
cor-
residues
the modified lysyl
are
has a single
of enolase
Similarly,
residues
residues
are
thought
not likely
(6).
phosphate, with
(2).
(4-6),
instance
acid
involved
site
residues
a substrate
a carboxyl yeast
group enolase
carbodiimides,
of carboxyl enolase
enolase.
the inactivation
to inactivate different
amino
residues
or catalysis
enolase
with
histidyl
mechanism.
glycidol
several
to the active
Although
(9),
that
which
of methionyl
conditions
shown
(l-3)
of substrates
to contain
in substrate
rabbit
subunit
the modification
to be involved
yeast
moiety
denaturing
involved
per
shown
indeed
1 Acknowledgement is made to the Donors administered by the American Chemical 2 Taken in part from the senior Independent of Wooster, 1978. 3 To whom inquiries should be addressed.
analogue, at the active (11).
reagents
residues
in proteins
has essential
inactivates
carboxyl
site
We have known
(10).
modified to be highly
(12-15).
Our
groups.
of the Petroleum Research Fund, Society, for support of this research. Study
thesis
of A. L. G.,
The
College
0006-291X/79/050059-07$01.00/0 59
Copyright @I 1979 by Academic Press. Inc. All rights of reproduction in anyform reserved.
8lOCHEMlCAL
Vol. 87, No. 1, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS 2 -Phosphoglycerate (2 -PGA)4, 3-PGA, EDC, CMC, and MES were obtained from Sigma. Methyl iodide was purchased from Matheson, Coleman, and Bell, and G-6-P was from Boehringer Mannheim. Yeast enolase, from Sigma, was characterized and assayed as previously described (2). EAC was synthesized from EDC and methyl iodide by published procedures (16). Chemical modifications of enolase were carried out at 25’ under the conditions given in the figure and table legends. Modifications were usually initiated by the addition of a freshly prepared stock solution of carbodiimide in buffer to a solution of enzyme, in some cases together with substrates, inhibitors, or G-6-P, in the same buffer. In some experiments, carbodiimides were pre-incubated in buffer with substrates, inhibitors, or G-6-P for various times before initiating the modification by addition of enzyme in order to determine the effect of phosphorylated compounds on the carbodiimide. Aliquots were periodically withdrawn, assayed, and compared to a control subjected to the same conditions but in the absence of carbodiimide. In some cases, aliquots were passed through a column (0.9 x 40 cm) of Bio-Gel p-6 to remove excess reagents, assayed for specific activity, and hydrolyzed for 18 hr in 6 N HCl at 105O and subjected to amino acid analysis on a Beckman 120C amino acid analyzer. RESULTS Yeast
enolase
inactivation
is rapidly
markedly
enolase
is modified
EDTA,
pH 6.1,
CMC,
in that
9 min
with
Although residues
Under
29 min
in proteins
at slightly
teine. are
stable
65% inactivated 4
reagents
it is very
unlikely
that
cysteine.
This
(9),
Tyrosine very
modifying
conditions
can also to acid by EDC
react
hydrolysis
was with
and tyrosine
inactivation
is supported
one under
by carbodiimides by the fact
0-arylisoureas
analysis
of enolase
of tyrosine
only
except
to form
no loss
after
has
carbodiimides
at pH 6.1 shows
and
can also
shows
acid
mM
of carboxyl
by EDC
Amino
0.01
by EDC
95% inactivated
(17).
When
MgC12,
the modification
towards
these
relative
no loss
that
amino
of cyswhich which
was
to the control,
Abbreviations used are: PGA, phosphoglycerate; MES, 2-(N-morpholino)G-6-P, glucose-6-phosphate; Pi, inorganic phosphate; ethanesulfonic acid; EDC, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride; CMC, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate; EAC, l-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide (also known by l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide methiodide, a name less descriptive of the actual structure of the compound).
60
of
CMC.
Enolase
is unreactive
which
for
1).
is achieved
(13,17).
which
of enolase
with
the rate
followed
conditions
subunit
of this
agent,
cysteine
under
thus
1 mM
pH (12,13),
extent
analysis
selective
acidic
MES,
with
(Figure
70% inactivation
and 56 min
highly
and EAC,
employed
inactivating
conditions,
EDC, are
is due to the modification acid
these
with
EDC,
in 50 mM
effective
to some per
denaturing
carbodiimide
is the most
order.
by CMC,
on the carbodiimide
by 20 mM
carbodiimides
be modified cysteine
dependent
EAC
EAC,
inactivated
BIOCHEMICAL
Vol. 87, No. 1, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MINUTES inactivation FIGURE 1: Yeast enolase was modified by 20 mM carbodiimide mM EDTA, pH 6.1. The carbodiimides and (A ) CMC. The control retains experiment.
while
a sample
which
(data
not shown).
was
95% inactivated
These
data
is due to the modification that
carbodiimides The
0-acylisourea
N-acylurea,
undergo
transfer
indicated
by our
reagent
is removed
observation
for
cinamide
hydrochloride
All
the formation
of
contain
imides
react
protection
both
for
of enolase,
phosphate
(Table
I).
under
these
little
with
conditions.
and carboxylate
competitive
functionalities
enolase
be interpreted
at pH 6.1 with
of competitive However,
that
either
with
3-PGA
or Pi,
is observed
after
inhibitors,
enolase
protection
61
may
M,
EDC
only
excess
Further
that neither
gly-
cause
any
at pH 6.1. inhibitors
(8).
of organic
is
then
3 hr.
at 0.4
by 20 mM
to
N-acylurea
within
observation
moieties
an
stable
by EDC,
occurs
and carboxyl must
to form
of a stable
of Pi (Z),
suggest
or hydrolyze
hydrochloride,
the exception
or no inactivation
In the absence
nucleophile,
of enolase
by EDC
to a more
is 95% inactivated
semicarbazide
phosphate
45 min
amine
is our
subunit
inactivation
is thought
rearrange
no reactivation
of inactivation with
experiments
pre-incubated tors
nor
and,
with
if enolase
per
residues.
either
of an N-acylurea
the rate
substrates
enolase
that
tyr
and strongly
groups
The formation
(12,13).
by gel filtration,
evidence
acceleration
can then
that
carboxyl
carboxyl
to a suitable
group
0.2 less
residues,
by modifying
with
which
the carboxyl
or tyrosyl
enolase
of carbodiimides
only
the possibility
of cysteinyl
intermediate
regenerate
shows
eliminate
inactivate
reaction
by carbodiimides. Enolase, l&M, in 50 mM MES, 1 mM MgCl , 0.01 used were (0) EAC, ( d 1 EDC, full activity for the duration of the
of
Since
carbodi-
compounds care.
(18,19),
If EAC
competitive
is
inhibi-
1 hr of modification is 99% inactivated be apparent
and is
BIOCHEMICAL
Vol. 87, No. 1, 1979
TABLE
I:
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect
of Phosphorylated
Compounds
Yeast
Enolase
Effect
Added
Compound
by EAC.
on the Inactivation
of
of Pre -incubationa ‘$J Inhibition
None
3-PGA G-6-P aEAC, 25 mM, was pre incubated for 45 min with either 125 mM Pit 25 mM 3-PGA, or 25 mM G-6-P in 50 mM MES, 1 mM MgC12, 0.01 mM EDTA, pH 6.1. Modification was then initiated by addition of an aliquot of enzyme in 0.25 volume of buffer, giving final concentrations of 1JlM enolase, 20 mM FAC, 20 mM 3-PGA or G-6-P, and 100 mM Pi. After 1 hr an aliquot was assayed for enzyme activity relative to a control subjected to the same conditions but in the absence of EAC.
TABLE
II:
Effect
of 3-PGA
Effect
of Time
Time
on the Inactivation
of Yeast
of Pre-incubation
of Pre-incubation,
of EAC
min
Enolase
with
by EAC.
3-PGAa
$I Activity
0
32
10
58
20
82
30
100
aConditions the same as in Table I, except that EAC was pre-incubated with 3-PGA for the indicated time before initiating modification by Enzyme activity was determined after 30 min modiadding enzyme. fication.
likely
due to the reaction
pounds
with
agent,
is suggested
also
carbodiimide,
provides Table
almost
complete
II suggests
the extent
of inactivation
for
0, 10, 20,
is modified
and 30 min
before
which against
EAC
the addition
62
of these
concentration
com-
of modifying of enolase,
inactivation
I).
with
(Table 3-PGA
in the presence
on the order 3-PGA.
moities
is not an inhibitor
of carbodiimides
by 20 mM
with
carboxyl
the effective
G-6-P,
protection
is dependent of EAC
and/or
reducing
that the reaction
If enolase
of pre-incubation
thus
by the fact that
pendent.
time
of the phosphate
If EAC
of addition and 3-PGA
of enzyme,
activity
is time
of 20 mM of reagents are
de3-PGA, and the
pre-incubated
after
30 min
BIOCHEMICAL
Vol. 87, No. 1, 1979
04 0
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6
MINZTES
against F.AC inactivation of yeast enolase. Enolase, FIGURE 2: Protection l@, in 50 mM MES, 1 mM MgC12, 0.01 mM EDTA, pH 6.1, was modified by 40 mM EAC, either in the absence (0) of added phosphorylated compounds, or in the presence of (m) 40 mM G-6-P plus 40 mM acetate, or 40 mM 3-PGA, or ( + ) 40 mM I-PGA. In each ( A) 40 mM Pit or (v) instance, the modification was initiated by adding an aliquot of a freshlyprepared stock solution of EAC to the enzyme plus phosphorylated compound.
modification gests
is 32$&, 58%,
that
protection
experiments
and concentrations results
acetate, Table
an experiment
EAC
at pH 6.1,
40 mM I).
each,
Pi provides
and substrates tation
data
carbodiimides active
be possible
are activity
shown
no protection
a small
amount
the greatest
is difficult,
is likely
(inhibitor)
in Figure
is reduced
are
to 10% after
of protection.
it suggests
due to the modification
The
to
more
exact
of yeast
protection,
interpre
enolase
residues
by
plus
(compare affords
of carboxyl
brief
G-6-P
While
that inactivation
are
is inactivated
6 min.
3-PGA
sug-
comparable.
inactivation
of protection,
This times
If enolase
2.
against
degree
respectively.
if modification
and substrate
provide
provide
of this
may
of carbodiimide
of such
by 40 mM
82oJ0, and 100% of the control,
-
by
at the enzyme
site.
DISCUSSION The
data
suggest
that
yeast
Z-PGA
affords
the greatest
imides
(Figure
2), it is likely
groups
at the active
tive
by our
observation
inhibitor,
affords
However,
this
moiety
with
site
enolase
degree
has essential
of protection
that inactivation
of the enzyme. that
significant
protection
the carbodiimide,
G-6-P, protection
by G-6-P thus
against
inactivation
conclusion
is neither under
is made
a substrate
the appropriate
Since
by carbodi-
due to the reaction
reducing
the effective
of carboxyl
somewhat nor
conditions
is likely
63
residues.
is due to the modification
This
which
carboxyl
tena-
competitive (Table
I).
of its phosphate
concentration
of modifying
Vol. 87, No. 1, 1979
agent.
A survey
undesirable
studies
(20,21).
substrates, rapidly
reduced
diimides
are
reported
possible philic
when
attack
moieties
Finally,
either
EAC
inactivates
be synthesized
that
EAC
may
against
or CMC
has been
by a carbodiimide.
in preliminary
form
enolase
much
coupled
with
in one step more
rapidly
the ease
with
for
further
which
commercially
protein
con-
and/or
by carbodiimides.
every
it very
report
of the
has been
reported
interesting
that
either EDC or CMC. This 14 the C -1abelled reagent available
modification
characterizing
with
to conditions
phosphate
use of EAC
than
it is
by a nucleo-
be given
containing
We find
more
(16) from
useful
currently
(25).
(24),
attached
must
them
substrates
attack
covalently
The
Carbo-
and activate
undergoes
in almost
be
is possible.
of enzymes
used
may
phosphorylated
attention
inactivation
prove
We are
that
of these
by carbodiimides
becoming
suggests
to protect
yeast
may
the substrate work
and inhibitors
reactivity,
or CMC.
of enzymes
of
inactivation
of substrate
where
substrate
concentrations
site
functionalities
in cases
modification of phos-
that
no protection
phosphate
Thus,
by high
concentration
to use substrates
EDC
and that
increased
with
for
concentrations
at the binding
where
to this
in protein
buffer
interpreted
residues
the inactivation
of an enzyme
once,
usually
to a level
(23).
Our
one attempts
only
are
low
inactivation
the effective
on the enzyme,
inactivation
results
to react
inactivation.
carboxyl
against
the carbodiimide-activated
group
comitant
that
of carboxyl
to accelerate
that
suggest
to protect
is given
carbodiimides
of studies
by carbodiimide
nucleophilic
with
use phosphate
Such
known
moieties
attention
studies
in actuallity
well
that little
several
modification
when
towards
fail
(20,22).
not involve
suggests
in fact,
substrates
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of phosphate
A number
carbodiimides
are
reaction
studies;
phorylated
does
of the literature
side
modification
BIOCHEMICAL
studies
EDC, than
the inactivation
suggests either
EDC
of enolase
by
EAC. Acknowledzements This chase
work
aided
of the Beckman
out during made
was
120C amino
the summer
possible
by a grant
from acid
the Research analyzer.
of 1978 in an undergraduate
by Grant
SPI77-25616
from
Corporation
Parts
of this
research
the National
for work
the purwere
participation
Science
carried program
Foundation.
REFERENCES 1.
Riordan, J. F., 195, 884-886.
McElvany,
K.D.
and Borders,
64
C. L.,
Jr.
(1977)
Science,
Vol. 87, No. 1, 1979
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Borders, C.L., Jr., Woodall, M. L. and George, A. L., Jr. (1978) Biochem. Biophys. Res. Comm., 82, 901-906. Elliott, J. I. and Brewer, J. M. (1978) Arch. Biochem. Biophys., 190, 351-357. Westhead, E. W. (1965) Biochemistry, 4, 2139-2144. Elliott, J.I. and Brewer, J.M. (1978) Fed. Proc., 37, 1813. George, A. L., Jr. and Borders, C. L., Jr. (1979) submitted for publication. Brake, J.M. and Wold, F. (1962) Biochemistry, 1, 386-391. Wold, F. (1971) The Enzymes, 3rd Ed., 5, 499-538. Oh, S. -K., Travis, J. and Brewer, J.M. (1973) Biochim. Biophys. Acta, 310, 421-429. Schray, K. J., O’Connell, E. L. and Rose, I.A. (1973) J. Biol. Chem., 248, 2214-2218. Shen, T. Y.S. and Westhead, E. W. (1973) Biochemistry, 12, 3333-3337. Hoare, D. G. and Koshland, D.E., Jr. (1967) J. Biol. Chem., 242, 2447-2453. Carraway, K. L. and Koshland, D.E., Jr. (1972) Methods Enzymol., 25, 616-623. Lin, T. -Y. and Koshland, D.E., Jr. (1969) J. Biol. Chem., 244, 505-508. Riordan, J. F. and Hayashida, H. (1970) Biochem. Biophys. Res. Comm., 41, 122-127. Sheehan, J. C. Cruickshank, P.A. and Boshart, G. L. (1961) J. Org. Chem., 26, 2525-2528. Carraway, K. L. and Koshland, D. E., Jr. (1968) Biochim. Biophys. Acta, 160, 272-274. Khorana, H. G. (1953), Chem. Rev. , 53, 145 -166. Kurzer, F. and Douraghi-Zadeh, K. (1967) Chem. Rev., 67, 107-152. Hut. C., Olomucki, A. and Thorn;-Beau, F. (1975) F.E. B.S. Lett., 60, 414-418. Pho, D. B., Roustan, C., Tot, A. N. T. and Pradel, L. -A. (1977) Biochemistry, 16, 4533-4537. Swaisgood, H. and Natake, M. (1973) J, Biochem. (Tokyo), 74, 77-86. Khorana, H. G. (1961) Some Recent Developments in the Chemistry of Phosphate Esters of Biological Interest, John Wiley and Sons, New York. Ariki, M. Fukui, T. (1978) J. Biochem. (Tokyo), 83, 183-190. Timkovich, R. (1977) Anal. Biochem., 79, 135-143.
65
Vol. 87, No. 1, 1979 March.15,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
1979
ESSENTIAL
CARBOXY
Alfred Department Received
L.
George,
of Chemistry,
January
L RESIDUES 2
Jr.
IN YEAST
and C.
College
L.
ENOLASE’
Borders,
of Wooster,
59-65
3
Jr.
Wooster,
Ohio
44691
19,1979
Yeast enolase (EC 4.2.1.11) is rapidly inactivated at pH 6.1 by three different water-soluble carbodiimides -- 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, and l-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide. Inactivation is most likely due to the modification of essential carboxyl residues at the enzyme active site. Chemical essential
for
modification
studies
have
the catalytic
activity
of yeast
sential
arginyl
residue
anionic
phosphate
has been
shown
histidine
is less
related
with
under
It has been muscle
However,
selective study
well
essential
defined.
in the catalytic
this
enolase for suggests
binding that
by reaction
reagent
fails
several
the modification that
yeast
The
enzyme
is likely
in neither
(7,8) are
residues
in binding
enolase played
has been
and cysteinyl
es-
the
In addition, but the role
by
cor-
residues
the modified lysyl
are
has a single
of enolase
Similarly,
residues
residues
are
thought
not likely
(6).
phosphate, with
(2).
(4-6),
instance
acid
involved
site
residues
a substrate
a carboxyl yeast
group enolase
carbodiimides,
of carboxyl enolase
enolase.
the inactivation
to inactivate different
amino
residues
or catalysis
enolase
with
histidyl
mechanism.
glycidol
several
to the active
Although
(9),
that
which
of methionyl
conditions
shown
(l-3)
of substrates
to contain
in substrate
rabbit
subunit
the modification
to be involved
yeast
moiety
denaturing
involved
per
shown
indeed
1 Acknowledgement is made to the Donors administered by the American Chemical 2 Taken in part from the senior Independent of Wooster, 1978. 3 To whom inquiries should be addressed.
analogue, at the active (11).
reagents
residues
in proteins
has essential
inactivates
carboxyl
site
We have known
(10).
modified to be highly
(12-15).
Our
groups.
of the Petroleum Research Fund, Society, for support of this research. Study
thesis
of A. L. G.,
The
College
0006-291X/79/050059-07$01.00/0 59
Copyright @I 1979 by Academic Press. Inc. All rights of reproduction in anyform reserved.
8lOCHEMlCAL
Vol. 87, No. 1, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS 2 -Phosphoglycerate (2 -PGA)4, 3-PGA, EDC, CMC, and MES were obtained from Sigma. Methyl iodide was purchased from Matheson, Coleman, and Bell, and G-6-P was from Boehringer Mannheim. Yeast enolase, from Sigma, was characterized and assayed as previously described (2). EAC was synthesized from EDC and methyl iodide by published procedures (16). Chemical modifications of enolase were carried out at 25’ under the conditions given in the figure and table legends. Modifications were usually initiated by the addition of a freshly prepared stock solution of carbodiimide in buffer to a solution of enzyme, in some cases together with substrates, inhibitors, or G-6-P, in the same buffer. In some experiments, carbodiimides were pre-incubated in buffer with substrates, inhibitors, or G-6-P for various times before initiating the modification by addition of enzyme in order to determine the effect of phosphorylated compounds on the carbodiimide. Aliquots were periodically withdrawn, assayed, and compared to a control subjected to the same conditions but in the absence of carbodiimide. In some cases, aliquots were passed through a column (0.9 x 40 cm) of Bio-Gel p-6 to remove excess reagents, assayed for specific activity, and hydrolyzed for 18 hr in 6 N HCl at 105O and subjected to amino acid analysis on a Beckman 120C amino acid analyzer. RESULTS Yeast
enolase
inactivation
is rapidly
markedly
enolase
is modified
EDTA,
pH 6.1,
CMC,
in that
9 min
with
Although residues
Under
29 min
in proteins
at slightly
teine. are
stable
65% inactivated 4
reagents
it is very
unlikely
that
cysteine.
This
(9),
Tyrosine very
modifying
conditions
can also to acid by EDC
react
hydrolysis
was with
and tyrosine
inactivation
is supported
one under
by carbodiimides by the fact
0-arylisoureas
analysis
of enolase
of tyrosine
only
except
to form
no loss
after
has
carbodiimides
at pH 6.1 shows
and
can also
shows
acid
mM
of carboxyl
by EDC
Amino
0.01
by EDC
95% inactivated
(17).
When
MgC12,
the modification
towards
these
relative
no loss
that
amino
of cyswhich which
was
to the control,
Abbreviations used are: PGA, phosphoglycerate; MES, 2-(N-morpholino)G-6-P, glucose-6-phosphate; Pi, inorganic phosphate; ethanesulfonic acid; EDC, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride; CMC, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate; EAC, l-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide (also known by l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide methiodide, a name less descriptive of the actual structure of the compound).
60
of
CMC.
Enolase
is unreactive
which
for
1).
is achieved
(13,17).
which
of enolase
with
the rate
followed
conditions
subunit
of this
agent,
cysteine
under
thus
1 mM
pH (12,13),
extent
analysis
selective
acidic
MES,
with
(Figure
70% inactivation
and 56 min
highly
and EAC,
employed
inactivating
conditions,
EDC, are
is due to the modification acid
these
with
EDC,
in 50 mM
effective
to some per
denaturing
carbodiimide
is the most
order.
by CMC,
on the carbodiimide
by 20 mM
carbodiimides
be modified cysteine
dependent
EAC
EAC,
inactivated
BIOCHEMICAL
Vol. 87, No. 1, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MINUTES inactivation FIGURE 1: Yeast enolase was modified by 20 mM carbodiimide mM EDTA, pH 6.1. The carbodiimides and (A ) CMC. The control retains experiment.
while
a sample
which
(data
not shown).
was
95% inactivated
These
data
is due to the modification that
carbodiimides The
0-acylisourea
N-acylurea,
undergo
transfer
indicated
by our
reagent
is removed
observation
for
cinamide
hydrochloride
All
the formation
of
contain
imides
react
protection
both
for
of enolase,
phosphate
(Table
I).
under
these
little
with
conditions.
and carboxylate
competitive
functionalities
enolase
be interpreted
at pH 6.1 with
of competitive However,
that
either
with
3-PGA
or Pi,
is observed
after
inhibitors,
enolase
protection
61
may
M,
EDC
only
excess
Further
that neither
gly-
cause
any
at pH 6.1. inhibitors
(8).
of organic
is
then
3 hr.
at 0.4
by 20 mM
to
N-acylurea
within
observation
moieties
an
stable
by EDC,
occurs
and carboxyl must
to form
of a stable
of Pi (Z),
suggest
or hydrolyze
hydrochloride,
the exception
or no inactivation
In the absence
nucleophile,
of enolase
by EDC
to a more
is 95% inactivated
semicarbazide
phosphate
45 min
amine
is our
subunit
inactivation
is thought
rearrange
no reactivation
of inactivation with
experiments
pre-incubated tors
nor
and,
with
if enolase
per
residues.
either
of an N-acylurea
the rate
substrates
enolase
that
tyr
and strongly
groups
The formation
(12,13).
by gel filtration,
evidence
acceleration
can then
that
carboxyl
carboxyl
to a suitable
group
0.2 less
residues,
by modifying
with
which
the carboxyl
or tyrosyl
enolase
of carbodiimides
only
the possibility
of cysteinyl
intermediate
regenerate
shows
eliminate
inactivate
reaction
by carbodiimides. Enolase, l&M, in 50 mM MES, 1 mM MgCl , 0.01 used were (0) EAC, ( d 1 EDC, full activity for the duration of the
of
Since
carbodi-
compounds care.
(18,19),
If EAC
competitive
is
inhibi-
1 hr of modification is 99% inactivated be apparent
and is
BIOCHEMICAL
Vol. 87, No. 1, 1979
TABLE
I:
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect
of Phosphorylated
Compounds
Yeast
Enolase
Effect
Added
Compound
by EAC.
on the Inactivation
of
of Pre -incubationa ‘$J Inhibition
None
3-PGA G-6-P aEAC, 25 mM, was pre incubated for 45 min with either 125 mM Pit 25 mM 3-PGA, or 25 mM G-6-P in 50 mM MES, 1 mM MgC12, 0.01 mM EDTA, pH 6.1. Modification was then initiated by addition of an aliquot of enzyme in 0.25 volume of buffer, giving final concentrations of 1JlM enolase, 20 mM FAC, 20 mM 3-PGA or G-6-P, and 100 mM Pi. After 1 hr an aliquot was assayed for enzyme activity relative to a control subjected to the same conditions but in the absence of EAC.
TABLE
II:
Effect
of 3-PGA
Effect
of Time
Time
on the Inactivation
of Yeast
of Pre-incubation
of Pre-incubation,
of EAC
min
Enolase
with
by EAC.
3-PGAa
$I Activity
0
32
10
58
20
82
30
100
aConditions the same as in Table I, except that EAC was pre-incubated with 3-PGA for the indicated time before initiating modification by Enzyme activity was determined after 30 min modiadding enzyme. fication.
likely
due to the reaction
pounds
with
agent,
is suggested
also
carbodiimide,
provides Table
almost
complete
II suggests
the extent
of inactivation
for
0, 10, 20,
is modified
and 30 min
before
which against
EAC
the addition
62
of these
concentration
com-
of modifying of enolase,
inactivation
I).
with
(Table 3-PGA
in the presence
on the order 3-PGA.
moities
is not an inhibitor
of carbodiimides
by 20 mM
with
carboxyl
the effective
G-6-P,
protection
is dependent of EAC
and/or
reducing
that the reaction
If enolase
of pre-incubation
thus
by the fact that
pendent.
time
of the phosphate
If EAC
of addition and 3-PGA
of enzyme,
activity
is time
of 20 mM of reagents are
de3-PGA, and the
pre-incubated
after
30 min
BIOCHEMICAL
Vol. 87, No. 1, 1979
04 0
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6
MINZTES
against F.AC inactivation of yeast enolase. Enolase, FIGURE 2: Protection l@, in 50 mM MES, 1 mM MgC12, 0.01 mM EDTA, pH 6.1, was modified by 40 mM EAC, either in the absence (0) of added phosphorylated compounds, or in the presence of (m) 40 mM G-6-P plus 40 mM acetate, or 40 mM 3-PGA, or ( + ) 40 mM I-PGA. In each ( A) 40 mM Pit or (v) instance, the modification was initiated by adding an aliquot of a freshlyprepared stock solution of EAC to the enzyme plus phosphorylated compound.
modification gests
is 32$&, 58%,
that
protection
experiments
and concentrations results
acetate, Table
an experiment
EAC
at pH 6.1,
40 mM I).
each,
Pi provides
and substrates tation
data
carbodiimides active
be possible
are activity
shown
no protection
a small
amount
the greatest
is difficult,
is likely
(inhibitor)
in Figure
is reduced
are
to 10% after
of protection.
it suggests
due to the modification
The
to
more
exact
of yeast
protection,
interpre
enolase
residues
by
plus
(compare affords
of carboxyl
brief
G-6-P
While
that inactivation
are
is inactivated
6 min.
3-PGA
sug-
comparable.
inactivation
of protection,
This times
If enolase
2.
against
degree
respectively.
if modification
and substrate
provide
provide
of this
may
of carbodiimide
of such
by 40 mM
82oJ0, and 100% of the control,
-
by
at the enzyme
site.
DISCUSSION The
data
suggest
that
yeast
Z-PGA
affords
the greatest
imides
(Figure
2), it is likely
groups
at the active
tive
by our
observation
inhibitor,
affords
However,
this
moiety
with
site
enolase
degree
has essential
of protection
that inactivation
of the enzyme. that
significant
protection
the carbodiimide,
G-6-P, protection
by G-6-P thus
against
inactivation
conclusion
is neither under
is made
a substrate
the appropriate
Since
by carbodi-
due to the reaction
reducing
the effective
of carboxyl
somewhat nor
conditions
is likely
63
residues.
is due to the modification
This
which
carboxyl
tena-
competitive (Table
I).
of its phosphate
concentration
of modifying
Vol. 87, No. 1, 1979
agent.
A survey
undesirable
studies
(20,21).
substrates, rapidly
reduced
diimides
are
reported
possible philic
when
attack
moieties
Finally,
either
EAC
inactivates
be synthesized
that
EAC
may
against
or CMC
has been
by a carbodiimide.
in preliminary
form
enolase
much
coupled
with
in one step more
rapidly
the ease
with
for
further
which
commercially
protein
con-
and/or
by carbodiimides.
every
it very
report
of the
has been
reported
interesting
that
either EDC or CMC. This 14 the C -1abelled reagent available
modification
characterizing
with
to conditions
phosphate
use of EAC
than
it is
by a nucleo-
be given
containing
We find
more
(16) from
useful
currently
(25).
(24),
attached
must
them
substrates
attack
covalently
The
Carbo-
and activate
undergoes
in almost
be
is possible.
of enzymes
used
may
phosphorylated
attention
inactivation
prove
We are
that
of these
by carbodiimides
becoming
suggests
to protect
yeast
may
the substrate work
and inhibitors
reactivity,
or CMC.
of enzymes
of
inactivation
of substrate
where
substrate
concentrations
site
functionalities
in cases
modification of phos-
that
no protection
phosphate
Thus,
by high
concentration
to use substrates
EDC
and that
increased
with
for
concentrations
at the binding
where
to this
in protein
buffer
interpreted
residues
the inactivation
of an enzyme
once,
usually
to a level
(23).
Our
one attempts
only
are
low
inactivation
the effective
on the enzyme,
inactivation
results
to react
inactivation.
carboxyl
against
the carbodiimide-activated
group
comitant
that
of carboxyl
to accelerate
that
suggest
to protect
is given
carbodiimides
of studies
by carbodiimide
nucleophilic
with
use phosphate
Such
known
moieties
attention
studies
in actuallity
well
that little
several
modification
when
towards
fail
(20,22).
not involve
suggests
in fact,
substrates
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of phosphate
A number
carbodiimides
are
reaction
studies;
phorylated
does
of the literature
side
modification
BIOCHEMICAL
studies
EDC, than
the inactivation
suggests either
EDC
of enolase
by
EAC. Acknowledzements This chase
work
aided
of the Beckman
out during made
was
120C amino
the summer
possible
by a grant
from acid
the Research analyzer.
of 1978 in an undergraduate
by Grant
SPI77-25616
from
Corporation
Parts
of this
research
the National
for work
the purwere
participation
Science
carried program
Foundation.
REFERENCES 1.
Riordan, J. F., 195, 884-886.
McElvany,
K.D.
and Borders,
64
C. L.,
Jr.
(1977)
Science,
Vol. 87, No. 1, 1979
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Borders, C.L., Jr., Woodall, M. L. and George, A. L., Jr. (1978) Biochem. Biophys. Res. Comm., 82, 901-906. Elliott, J. I. and Brewer, J. M. (1978) Arch. Biochem. Biophys., 190, 351-357. Westhead, E. W. (1965) Biochemistry, 4, 2139-2144. Elliott, J.I. and Brewer, J.M. (1978) Fed. Proc., 37, 1813. George, A. L., Jr. and Borders, C. L., Jr. (1979) submitted for publication. Brake, J.M. and Wold, F. (1962) Biochemistry, 1, 386-391. Wold, F. (1971) The Enzymes, 3rd Ed., 5, 499-538. Oh, S. -K., Travis, J. and Brewer, J.M. (1973) Biochim. Biophys. Acta, 310, 421-429. Schray, K. J., O’Connell, E. L. and Rose, I.A. (1973) J. Biol. Chem., 248, 2214-2218. Shen, T. Y.S. and Westhead, E. W. (1973) Biochemistry, 12, 3333-3337. Hoare, D. G. and Koshland, D.E., Jr. (1967) J. Biol. Chem., 242, 2447-2453. Carraway, K. L. and Koshland, D.E., Jr. (1972) Methods Enzymol., 25, 616-623. Lin, T. -Y. and Koshland, D.E., Jr. (1969) J. Biol. Chem., 244, 505-508. Riordan, J. F. and Hayashida, H. (1970) Biochem. Biophys. Res. Comm., 41, 122-127. Sheehan, J. C. Cruickshank, P.A. and Boshart, G. L. (1961) J. Org. Chem., 26, 2525-2528. Carraway, K. L. and Koshland, D. E., Jr. (1968) Biochim. Biophys. Acta, 160, 272-274. Khorana, H. G. (1953), Chem. Rev. , 53, 145 -166. Kurzer, F. and Douraghi-Zadeh, K. (1967) Chem. Rev., 67, 107-152. Hut. C., Olomucki, A. and Thorn;-Beau, F. (1975) F.E. B.S. Lett., 60, 414-418. Pho, D. B., Roustan, C., Tot, A. N. T. and Pradel, L. -A. (1977) Biochemistry, 16, 4533-4537. Swaisgood, H. and Natake, M. (1973) J, Biochem. (Tokyo), 74, 77-86. Khorana, H. G. (1961) Some Recent Developments in the Chemistry of Phosphate Esters of Biological Interest, John Wiley and Sons, New York. Ariki, M. Fukui, T. (1978) J. Biochem. (Tokyo), 83, 183-190. Timkovich, R. (1977) Anal. Biochem., 79, 135-143.
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