Vol.
86,
No.
January
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 387-394
30, 1979
EVIDENCE FOR AN ESSENTIAL
LYSINE RESIDUE ON THE PHOSPHORIBOSYL TRANSFERASE
SUBUNIT OF THE ANTHRANILATE SYNTHETASE-ANTHRANILATE
5-PHOSPHORIBOSYL-
PYROPHOSPHATE PHOSPHORIBOSYLTRANSFERASE ENZYME COMPLEX FROM SALMONELLA TYPHIMURIUM
Thomas H. Grove* and H. Richard Levy Research Laboratories, Department of Biology, Syracuse University, Syracuse, N.Y. 13210
Biological
Received
December
14,
1978
SUMMARY Pyridoxal 5'-phosphate reacts with the anthranilate synthetase-phosphoribosyltransferase enzyme complex of Salmonella typhimurium to inhibit PR transferase activity. Glutamine-dependent anthranilate synthetase is not affected. Spectral and kinetic data suggest that the inactivation results from the modification of an essential lysine residue which interacts with 5-phosphoribosyl 1-pyrophosphate. The anthranilate catalyzes
the
synthetase?
first
enzyme complex
two reactions
of tryptophan
units
each of AS and PRT which
comprise
sites
for
a divalent
AS);
glutamine
chorismate,
ammonia,
amino
acids
except
for
glutamine
concerned evidence
*
that
to AS-PRT from
amidotransferases evidence
with
that
(2,
metal portion
portion
binding a cysteine
residue
an essential
lysine
(1) therein).
residue
Present address: Department of Biochemistry, Parks Road, Oxford OX1 3QU, Great Britain.
t The abbreviations used are: late 5-phosphoribosylpyrophosphate phosphoribosyl 1-pyrophosphate; pyridoxamine 5'-phosphate.
is
is
specific
and tryptophan
of PRT);
ligands
typhimurium The two sub-
carry
ion,
of PRT).
these
S. typhimurium
and references
biosynthesis.
the complex
(on the amino-terminal
and PRPP (on the carboxy-terminal
from Salmonella
is
at their involved
known about
specific in binding
and to other,
analogous
The present
study
at the active
University
(on
and anthranilate
Little
present
binding
of Oxford,
AS = anthranilate synthetase; phosphoribosyltransferase; PLP = pyridoxal 5'-phosphate;
the
sites of glutamine provides site
on
South
PRT = anthraniPRPP = 5PMP =
0006-291X/79/020387-08$01.00/0 387
Copyright All rights
@ 1979 by Acudemic Press. Inc. of reproduction in anyfkn re.wrwd.
Vol. 86, No. 2, 1979
BIOCHEMICAL
the carboxy-terminal
portion
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of PRT and that
it
probably
functions
to bind
PRPP or Mg+'-PRPP. MATERIALS
AND METHODS
The preparation of the enzyme complex from 5. typhimurium has been described (3). Glutamine-dependent AS activity was measured by the method of Henderson --et al (4). PRT was assayed spectrophotometrically (3) on a Gilford 240 spectrophotometer at 320 nm in 20 mM barbitol, pH 8.9. Protein concentration was determined using the Lowry procedure. Incubations with PLP were carried out at O-4" in the dark in 50 mM barbitol, pH 8.7. Control incubations received water instead of PLP. There was a 5-15% loss of PRT activity in 25-30 min. in these control incubations; assays of PLP-treated enzyme were corrected for these non-specific activity losses. A buffer used extensively in these studies, referred to as Buffer 1 in the text, had the following composition: 0.05 M potassium phosphate, 0.1 mM EDTA, 0.4 mM 2-mercaptoethanol, 30% (v/v) glycerol, pH 7.4. Barium chorismate, PRPP and L-glutamine were obtained from Sigma Chemical Company; chorismate was converted to the potassium salt before use. Anthranilate (once recrystallized) came from Eastman Chemical Company and PLP from Nutritional Biochemicals Corporation. All other chemicals were of reagent grade.
RESULTS When enzyme complex order,
time-dependent
AS activity
was incubated
loss
a reversible
modified
enzyme complex-PLP
stants, were against plot
k',
were
calculated log of the
gave a straight which
respect
equilibrium
using (PLP)
(6)
reciprocal
the
with
(5).
relationship
against
respect
Glutamine-dependent
leveling
off
in Fig.
with
(Fig. that
388
order
rate
of 0.87
log (Fig.
concentration
con(toes) (toe5 )-' 2).
A
of PLP (7)
min -1 and an x-intercept
from
3). PLP inhibited
Mgf+ and anthranilate to anthranilate
of activity
Plotting
a slope
the reciprocal of 0.07
after
1 and the half-times
to 5 = 0.693/k'. . line
declined
and non-covalently
The pseudo-first
a y-intercept
demonstrated
1).
covalently
the data
mM was calculated
studies
with
This
gave a straight
to PRPP at saturating
competitively
from
was a pseudo-first
mM PLP, PRT activity
between
adducts
of k'
line
(Fig.
activity.
determined
a Ki of 1.17 Inhibition
At 1.89
initial
reflects
PLP, there
of PRT activity
was not affected.
26 min to 35% of the
with
competitively (Ki
at saturating
with
= 0.44 mM), and non++ Mg and non-
BIOCHEMICAL
Vol. 86, No. 2, 1979
TIME
;:g,.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(MINUTES)
1
Rate of inactivation of PRT activity with various concentrations of Enzyme complex (206 pg/ml) was incubated in 50 mM barbitol, pH 8.7, with PLP'at 0.1 mM (A), 0.2 mM (0), 0.4 mM (m), 0.94 mM (a) and 1.89 mM (0). Aliquots were removed at the times indicated and assayed as described in Methods. V, is the initial velocity of the control at zero time, Vi represents the initial velocity of the test sample at various times during the incubation.
-1.0
-0.8
-0.6 LOG
-0.4 [PYRIOOXAL
-0.2
0
0.2
C-P]
Fig. 2 Determination of the order of PLP inhibition of PRT activity. for PLP were taken from Fig. 1 and plotted as indicated in the text. centrations are molar; half-times are in min.
389
Data Con-
Vol.
86, No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
[PYRIOOXAL
5’-p]
-’
RESEARCH
COMMUNICATIONS
mh4-’
Fig. 3 Double reciprocal plot of the pseudo-first order rate constant PLP inhibition of PRT activity versus concentration of PLP. Data for were taken from Fig. 1 and plotted as indicated in the text. Table EFFECT
OF SUBSTRATES
for PLP
I
ON PLP INHIBITION
Additions
OF PRT ACTIVITY.a % Activity
Control
Remaining 100
None
42
PRPP
43
Anthranilate
48
Anthranilate
+ PRPP + EDTA
59
34
Mg+2 Mg+2
+ anthranilate
39
Mg+2
+ PRPP
74
aEnzyme complex (144 ug/ml) was incubated in 50 mM barbitol, pH 8.7, at 0-4°C in the dark. PLP was present at 1.89 mM in all of the incubations except for the control which contained none. The concentrations of Mg+2, PRPP, anthranilate and EDTA were 5 mM (38 Km), 1.26 mM (42 Km), 1.40 mM (117 Km) and 2 mM, respectively. The % activity remaining after 26 min. is expressed relative to the control.
saturating against
PRPP. PLP
afforded inhibition
inhibition
any significant slightly.
Incubation inhibition tion
with
Studies
the
showed
mM PLP.
protection
that
only
protection
the
1).
activity
0.98
20 hours
by of
Mgf+ alone
was found
with for
PRT
combination
enzyme complex
incubation Dialysis
of
(Table
Free anthranilate
of PLP-inhibited
by 60% after 1.96
on
not
Mgft
substrates and
PRPP
enhanced
PLP
to react
in 50 mM Tris
with reversed
mM PLP and by 45% after against
Buffer
PLP.
incuba-
1 completely
Vol.
86,
No.
BIOCHEMICAL
2, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1
0.10 0.08
8
0.06
z 2
0.04
2 Q
0.02 0 -0.02 /
/
300
350
I
/ 450
I 400
WAVELENGTH
(nm)
Fig. 4 Difference spectrum of the enzyme complex-PLP reversible adduct. The buffer used for all spectra was 0.05 M potassium phosphate, pH 7.4. The spectrum of enzyme complex (515 Fig/ml) in the presence of 0.4 mM PLP was measured 26 min after the addition of PLP. The blank was buffer containing 0.4 mM PLP. The difference spectrum was generated by subtracting from the above spectrum the spectrum of 515 vg/ml enzyme complex; the blank for this spectrum was buffer. Spectra were determined with a Cary 15 spectrophotometer.
reversed of
a primary
The is
inhibition.
This
amine
difference shown
In
(5,8),
Fig.
to
complex
was
incubated
dialyzed
for
at
20
number
hours
activity enzyme
for
moles was
with in
the
coefficients
nm (12) of
between
determine
extinction 324
not
is
consistent
a cysteine enzyme
or
complex
with
histidine and
PLP
modification
residue
(9,lO).
PLP-treated
enzyme
complex
4.
order
molar
but
spectrum
in
reversibility
of
inactivated,
the
stoichiometry
1.85
mM PLP
dark
of for
against
of
8700
26 min., volumes
cm-l
at
M-l
per
11 to
were mole
of
12 moles
complex.
391
enzyme of
bound
reduced
4000
N6-phosphopyridoxyllysine PMP bound
PMP groups
316 used
of
complex.
PMP were
with
bound
enzyme
NaBH
Buffer
nm (11) to
the
1. and
The
9700
determine When
70%
per
mole
and
4
M-'cm-'
the of
the of
Vol.
86,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
DISCUSSION PLP, a chemical shows high caused which
modification
selectivity
rapid
for
inhibition
the
of the
binding conjugates
after
PLP-enzyme
principally
to primary
330 nm (14,15).
two tautomeric
forms
enol-imine
spectrum
(Fig.
to primary
e-amino
mesenteroides isomerase
group (19)
which equal
tion
reacts to the
of PLP (6).
per active
unit
rate
of inactivation
(7).
Inactivation
a discrete Since the
of several evidence with average
groups
6-phosphate utilis
exist
form.
nm
The difference that
keto-enamine
the majority
It is
form.
bound reversibly from
and in rabbit
in
(16-18),-330
dehydrogenases
(20)
solution,
amines
suggests
when PLP is
PLP is
have an absorption
maxima
adduct
as the
found
complex
of the
PLP binds
order
to a
Leuconostoc
muscle
of the
of 0.87
were
portion
In Fig.
reaction
phosphoglucose
suggests
that
site
No subunit
PLP.
to infinite
PLP concentration
indicates to the
PLP.
This
a limiting
conversion suggests
392
of all that
line
concentra-
of PLP is bound
The data
for
of Kitz
phenomenon
interaction
of the
to the
one molecule
3 by the method
in Fig.
PLP and
of the PRT active
respect
inactivated.
to be a saturation
for
between
2, the slope
with
by PLP appears
owing with
analyzed
interaction
to that
of PRT when the enzyme is
the y-intercept min-'),
aspects
that
The slope
binding
origin,
mediate
complex
difference
aqueous
keto-enamine
PRPP or Mg+2-PRPP.
the extrapolation
k = (0.07
the
by PLP,
that
of primary
absorption
(13),
(21).
AS-PRT provides
is
in glucose and ___Candida
An examination
site
spectra
evidence
and
residues
inhibition
In neutral
PLP conjugates
amino
lysine
or tryptophan
and 430 nm for
to the difference
lysine
provide
histidine
nucleophiles
NaBH4, and the
groups.
4) of the PLP-enzyme
with
of specific
with
characteristic
form
of PLP was bound similar
with
reacts
The reversible
adduct
amino
of PLP and cysteine,
the
group
reduction
complex
maximum around
for
e-amino
which
of PRT activity.
became irreversible
spectrum
reagent
the and Wilson
suggesting
is detectable. does not pass through
rate
of inactivation,
free
enzyme to an inter-
PLP is
substrate-like
and is
BIOCHEMICAL
Vol. 86, No. 2, 1979
not
inactivating
these
by bimolecular
data.
Inhibition
calculated
from
with
these
demonstrate respect
in Table
significant
1 show that
protection.
PRT activity
may actually
paramagnetic
resonance
the active
be with studies
only
This
the
site
and that
respect
which
reacts
groups
to the
groups,
reacts
+2 is Mn
with
e-amino
lysine
residues
PLP-modified
group
of lysine lysine
of Mg
t2 and for
ion requirement
for
PRPP.
bound
Electron
tightly
to
the
essential
could
There
which
in which
modified
is
uncertain.
amino
amino The close
selectivity
phosphate
a lysine
reacting
(19),
of PMP
The
be N-terminal
(23).
of
The primary acids
acids
and 72
similarity
and the spectra
PLP was shown to react
sugar
also
portion
as 11 to 12 moles
enzyme complex
and the remarkable bind
PLP is
are 4 N-terminal
of the PLP-modified
residues,
residue
PRT activity,
to that
of inactivation.
remains
in the enzyme complex
enzymes
residues
still
of lysine.
present
other
with
for
groups
the spectrum
with
of
the e-amino
of PLP in reacting suggests
strongly
that
residue.
ACKNOWLEDGEMENT This Health
work
was supported
by Grant
GM19839 from the
United
States
Public
Nagano, H., Zalkin, H., and Henderson, E. J. (1970), J. Biol. 245, 381 o-3820. Kawamura, M., Keim, P. S., Goto, Y., Zalkin, H., and Heinrikson, (1978), J. Biol. Chem., 253, 4659-4668. Grove, T. H., and Levy, H. R.T976), Biochim. Biophys. Acta., 464-474.
Chem.,
Service. REFERENCES
1. 2. 3.
to
mM is
the substrate
PRT by binding
PRPP or Mg"-PRPP.
with
residues
essential
and/or
between
that
substrate
on the AS-PRT enzyme complex
of the essential
amino
combination
the metal
bound per mole of enzyme even at a 70% level
identity
respect
A Ki of 0.44
suggests
have shown that
PLP apparently
additional
are
with
from
Kd = 12 UM (22).
In summary,
with
calculated
PLP is competitive
to anthranilate.
PRT is Mgf2 -PRPP and not PRPP alone,
PRPP, with
A Ki of 1.17 mM is
data.
The experiments PRPP afforded
kinetics.
s,tudies
PRPP and non-competitive
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
393
R. L. 5,
Vol.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
86,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Henderson, E. J . 3 Nagano, H., Zalkin, H., and Hwang, L. H. (1970), J. Biol. Chem., 245, 1416-1423. Biochem. J., 147, 351-358. Chen, S., and Engel, P. (1975), Levy, H. M., Leber. P. D.. and Ryan, E. M. (1963), J. Biol. Chem., "-238, 3654-3659. " Kitz, R., and Wilson, I. (1962), J. Biol. Chem., 237, 3245-3249. Anderson, B. M., Anderson, C. II., and Churchich, J. E. (1966), Biochemistry, 5, 2893-2900. Yost, F., and Harrison, J. (1971), Biochem. Biophys. Res. Commun., 42, 516-522. Rippa, M., and Pontremoli, S. (1969), Arch. Biochem. Biophys., 133, 112-118. R. B., Nolan, C., and Fischer, E. H. (1971), Forrey, A. W., Olsgaard, Biochimie., 3, 269-281. Chemical Modification of Means, G. E., and Feeney, R. E. (1971), Proteins, p. 134, Holden-Day, Inc., San Francisco. Colombo, G., and Marcus, F. (1974), Biochemistry, 13, 3085-3091. S., and Folkers, K. (1948), J. Am. Chem. Sot., 7D-, Heyl, D., Harris, 3429-3431. Buell, M. V., and Hansen, R. E. (1960), J. Am. Chem. Sot., 82, 6042-6049. J. Am. Chem. Sot., 79-, 485-490. Metzler, D. E. ,(1957), Heinert, D. and Martell, A. E. (1963), 3. Am. Chem. Sot., 85, 183-188. Morino, Y. and Snell, E. E. (1967), J. Biol. Chem., 242, 2800-2809. Milhausen, M. and Levy, H. R. (1975), Eur. J. Biochem., 3, 453-461. Hoppe-Seyler Z. Physiol. Chem., Domschke, W., and Domagk, G. F. (1969), 350, 1111-1116. Schnackerz, K. D., and Noltmann, E. A. (1971), Biochemistry, lo, 4837-4843. Robison, P. D., Nowak, T., and Levy, H. R. Arch. Biochem. Biophys. (in press). Henderson, E. J. and Zalkin, H. (1971), J. Biol. Chem., 246, 6891-6898.
394
86,
No.
January
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 387-394
30, 1979
EVIDENCE FOR AN ESSENTIAL
LYSINE RESIDUE ON THE PHOSPHORIBOSYL TRANSFERASE
SUBUNIT OF THE ANTHRANILATE SYNTHETASE-ANTHRANILATE
5-PHOSPHORIBOSYL-
PYROPHOSPHATE PHOSPHORIBOSYLTRANSFERASE ENZYME COMPLEX FROM SALMONELLA TYPHIMURIUM
Thomas H. Grove* and H. Richard Levy Research Laboratories, Department of Biology, Syracuse University, Syracuse, N.Y. 13210
Biological
Received
December
14,
1978
SUMMARY Pyridoxal 5'-phosphate reacts with the anthranilate synthetase-phosphoribosyltransferase enzyme complex of Salmonella typhimurium to inhibit PR transferase activity. Glutamine-dependent anthranilate synthetase is not affected. Spectral and kinetic data suggest that the inactivation results from the modification of an essential lysine residue which interacts with 5-phosphoribosyl 1-pyrophosphate. The anthranilate catalyzes
the
synthetase?
first
enzyme complex
two reactions
of tryptophan
units
each of AS and PRT which
comprise
sites
for
a divalent
AS);
glutamine
chorismate,
ammonia,
amino
acids
except
for
glutamine
concerned evidence
*
that
to AS-PRT from
amidotransferases evidence
with
that
(2,
metal portion
portion
binding a cysteine
residue
an essential
lysine
(1) therein).
residue
Present address: Department of Biochemistry, Parks Road, Oxford OX1 3QU, Great Britain.
t The abbreviations used are: late 5-phosphoribosylpyrophosphate phosphoribosyl 1-pyrophosphate; pyridoxamine 5'-phosphate.
is
is
specific
and tryptophan
of PRT);
ligands
typhimurium The two sub-
carry
ion,
of PRT).
these
S. typhimurium
and references
biosynthesis.
the complex
(on the amino-terminal
and PRPP (on the carboxy-terminal
from Salmonella
is
at their involved
known about
specific in binding
and to other,
analogous
The present
study
at the active
University
(on
and anthranilate
Little
present
binding
of Oxford,
AS = anthranilate synthetase; phosphoribosyltransferase; PLP = pyridoxal 5'-phosphate;
the
sites of glutamine provides site
on
South
PRT = anthraniPRPP = 5PMP =
0006-291X/79/020387-08$01.00/0 387
Copyright All rights
@ 1979 by Acudemic Press. Inc. of reproduction in anyfkn re.wrwd.
Vol. 86, No. 2, 1979
BIOCHEMICAL
the carboxy-terminal
portion
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of PRT and that
it
probably
functions
to bind
PRPP or Mg+'-PRPP. MATERIALS
AND METHODS
The preparation of the enzyme complex from 5. typhimurium has been described (3). Glutamine-dependent AS activity was measured by the method of Henderson --et al (4). PRT was assayed spectrophotometrically (3) on a Gilford 240 spectrophotometer at 320 nm in 20 mM barbitol, pH 8.9. Protein concentration was determined using the Lowry procedure. Incubations with PLP were carried out at O-4" in the dark in 50 mM barbitol, pH 8.7. Control incubations received water instead of PLP. There was a 5-15% loss of PRT activity in 25-30 min. in these control incubations; assays of PLP-treated enzyme were corrected for these non-specific activity losses. A buffer used extensively in these studies, referred to as Buffer 1 in the text, had the following composition: 0.05 M potassium phosphate, 0.1 mM EDTA, 0.4 mM 2-mercaptoethanol, 30% (v/v) glycerol, pH 7.4. Barium chorismate, PRPP and L-glutamine were obtained from Sigma Chemical Company; chorismate was converted to the potassium salt before use. Anthranilate (once recrystallized) came from Eastman Chemical Company and PLP from Nutritional Biochemicals Corporation. All other chemicals were of reagent grade.
RESULTS When enzyme complex order,
time-dependent
AS activity
was incubated
loss
a reversible
modified
enzyme complex-PLP
stants, were against plot
k',
were
calculated log of the
gave a straight which
respect
equilibrium
using (PLP)
(6)
reciprocal
the
with
(5).
relationship
against
respect
Glutamine-dependent
leveling
off
in Fig.
with
(Fig. that
388
order
rate
of 0.87
log (Fig.
concentration
con(toes) (toe5 )-' 2).
A
of PLP (7)
min -1 and an x-intercept
from
3). PLP inhibited
Mgf+ and anthranilate to anthranilate
of activity
Plotting
a slope
the reciprocal of 0.07
after
1 and the half-times
to 5 = 0.693/k'. . line
declined
and non-covalently
The pseudo-first
a y-intercept
demonstrated
1).
covalently
the data
mM was calculated
studies
with
This
gave a straight
to PRPP at saturating
competitively
from
was a pseudo-first
mM PLP, PRT activity
between
adducts
of k'
line
(Fig.
activity.
determined
a Ki of 1.17 Inhibition
At 1.89
initial
reflects
PLP, there
of PRT activity
was not affected.
26 min to 35% of the
with
competitively (Ki
at saturating
with
= 0.44 mM), and non++ Mg and non-
BIOCHEMICAL
Vol. 86, No. 2, 1979
TIME
;:g,.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(MINUTES)
1
Rate of inactivation of PRT activity with various concentrations of Enzyme complex (206 pg/ml) was incubated in 50 mM barbitol, pH 8.7, with PLP'at 0.1 mM (A), 0.2 mM (0), 0.4 mM (m), 0.94 mM (a) and 1.89 mM (0). Aliquots were removed at the times indicated and assayed as described in Methods. V, is the initial velocity of the control at zero time, Vi represents the initial velocity of the test sample at various times during the incubation.
-1.0
-0.8
-0.6 LOG
-0.4 [PYRIOOXAL
-0.2
0
0.2
C-P]
Fig. 2 Determination of the order of PLP inhibition of PRT activity. for PLP were taken from Fig. 1 and plotted as indicated in the text. centrations are molar; half-times are in min.
389
Data Con-
Vol.
86, No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
[PYRIOOXAL
5’-p]
-’
RESEARCH
COMMUNICATIONS
mh4-’
Fig. 3 Double reciprocal plot of the pseudo-first order rate constant PLP inhibition of PRT activity versus concentration of PLP. Data for were taken from Fig. 1 and plotted as indicated in the text. Table EFFECT
OF SUBSTRATES
for PLP
I
ON PLP INHIBITION
Additions
OF PRT ACTIVITY.a % Activity
Control
Remaining 100
None
42
PRPP
43
Anthranilate
48
Anthranilate
+ PRPP + EDTA
59
34
Mg+2 Mg+2
+ anthranilate
39
Mg+2
+ PRPP
74
aEnzyme complex (144 ug/ml) was incubated in 50 mM barbitol, pH 8.7, at 0-4°C in the dark. PLP was present at 1.89 mM in all of the incubations except for the control which contained none. The concentrations of Mg+2, PRPP, anthranilate and EDTA were 5 mM (38 Km), 1.26 mM (42 Km), 1.40 mM (117 Km) and 2 mM, respectively. The % activity remaining after 26 min. is expressed relative to the control.
saturating against
PRPP. PLP
afforded inhibition
inhibition
any significant slightly.
Incubation inhibition tion
with
Studies
the
showed
mM PLP.
protection
that
only
protection
the
1).
activity
0.98
20 hours
by of
Mgf+ alone
was found
with for
PRT
combination
enzyme complex
incubation Dialysis
of
(Table
Free anthranilate
of PLP-inhibited
by 60% after 1.96
on
not
Mgft
substrates and
PRPP
enhanced
PLP
to react
in 50 mM Tris
with reversed
mM PLP and by 45% after against
Buffer
PLP.
incuba-
1 completely
Vol.
86,
No.
BIOCHEMICAL
2, 1979
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1
0.10 0.08
8
0.06
z 2
0.04
2 Q
0.02 0 -0.02 /
/
300
350
I
/ 450
I 400
WAVELENGTH
(nm)
Fig. 4 Difference spectrum of the enzyme complex-PLP reversible adduct. The buffer used for all spectra was 0.05 M potassium phosphate, pH 7.4. The spectrum of enzyme complex (515 Fig/ml) in the presence of 0.4 mM PLP was measured 26 min after the addition of PLP. The blank was buffer containing 0.4 mM PLP. The difference spectrum was generated by subtracting from the above spectrum the spectrum of 515 vg/ml enzyme complex; the blank for this spectrum was buffer. Spectra were determined with a Cary 15 spectrophotometer.
reversed of
a primary
The is
inhibition.
This
amine
difference shown
In
(5,8),
Fig.
to
complex
was
incubated
dialyzed
for
at
20
number
hours
activity enzyme
for
moles was
with in
the
coefficients
nm (12) of
between
determine
extinction 324
not
is
consistent
a cysteine enzyme
or
complex
with
histidine and
PLP
modification
residue
(9,lO).
PLP-treated
enzyme
complex
4.
order
molar
but
spectrum
in
reversibility
of
inactivated,
the
stoichiometry
1.85
mM PLP
dark
of for
against
of
8700
26 min., volumes
cm-l
at
M-l
per
11 to
were mole
of
12 moles
complex.
391
enzyme of
bound
reduced
4000
N6-phosphopyridoxyllysine PMP bound
PMP groups
316 used
of
complex.
PMP were
with
bound
enzyme
NaBH
Buffer
nm (11) to
the
1. and
The
9700
determine When
70%
per
mole
and
4
M-'cm-'
the of
the of
Vol.
86,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
DISCUSSION PLP, a chemical shows high caused which
modification
selectivity
rapid
for
inhibition
the
of the
binding conjugates
after
PLP-enzyme
principally
to primary
330 nm (14,15).
two tautomeric
forms
enol-imine
spectrum
(Fig.
to primary
e-amino
mesenteroides isomerase
group (19)
which equal
tion
reacts to the
of PLP (6).
per active
unit
rate
of inactivation
(7).
Inactivation
a discrete Since the
of several evidence with average
groups
6-phosphate utilis
exist
form.
nm
The difference that
keto-enamine
the majority
It is
form.
bound reversibly from
and in rabbit
in
(16-18),-330
dehydrogenases
(20)
solution,
amines
suggests
when PLP is
PLP is
have an absorption
maxima
adduct
as the
found
complex
of the
PLP binds
order
to a
Leuconostoc
muscle
of the
of 0.87
were
portion
In Fig.
reaction
phosphoglucose
suggests
that
site
No subunit
PLP.
to infinite
PLP concentration
indicates to the
PLP.
This
a limiting
conversion suggests
392
of all that
line
concentra-
of PLP is bound
The data
for
of Kitz
phenomenon
interaction
of the
to the
one molecule
3 by the method
in Fig.
PLP and
of the PRT active
respect
inactivated.
to be a saturation
for
between
2, the slope
with
by PLP appears
owing with
analyzed
interaction
to that
of PRT when the enzyme is
the y-intercept min-'),
aspects
that
The slope
binding
origin,
mediate
complex
difference
aqueous
keto-enamine
PRPP or Mg+2-PRPP.
the extrapolation
k = (0.07
the
by PLP,
that
of primary
absorption
(13),
(21).
AS-PRT provides
is
in glucose and ___Candida
An examination
site
spectra
evidence
and
residues
inhibition
In neutral
PLP conjugates
amino
lysine
or tryptophan
and 430 nm for
to the difference
lysine
provide
histidine
nucleophiles
NaBH4, and the
groups.
4) of the PLP-enzyme
with
of specific
with
characteristic
form
of PLP was bound similar
with
reacts
The reversible
adduct
amino
of PLP and cysteine,
the
group
reduction
complex
maximum around
for
e-amino
which
of PRT activity.
became irreversible
spectrum
reagent
the and Wilson
suggesting
is detectable. does not pass through
rate
of inactivation,
free
enzyme to an inter-
PLP is
substrate-like
and is
BIOCHEMICAL
Vol. 86, No. 2, 1979
not
inactivating
these
by bimolecular
data.
Inhibition
calculated
from
with
these
demonstrate respect
in Table
significant
1 show that
protection.
PRT activity
may actually
paramagnetic
resonance
the active
be with studies
only
This
the
site
and that
respect
which
reacts
groups
to the
groups,
reacts
+2 is Mn
with
e-amino
lysine
residues
PLP-modified
group
of lysine lysine
of Mg
t2 and for
ion requirement
for
PRPP.
bound
Electron
tightly
to
the
essential
could
There
which
in which
modified
is
uncertain.
amino
amino The close
selectivity
phosphate
a lysine
reacting
(19),
of PMP
The
be N-terminal
(23).
of
The primary acids
acids
and 72
similarity
and the spectra
PLP was shown to react
sugar
also
portion
as 11 to 12 moles
enzyme complex
and the remarkable bind
PLP is
are 4 N-terminal
of the PLP-modified
residues,
residue
PRT activity,
to that
of inactivation.
remains
in the enzyme complex
enzymes
residues
still
of lysine.
present
other
with
for
groups
the spectrum
with
of
the e-amino
of PLP in reacting suggests
strongly
that
residue.
ACKNOWLEDGEMENT This Health
work
was supported
by Grant
GM19839 from the
United
States
Public
Nagano, H., Zalkin, H., and Henderson, E. J. (1970), J. Biol. 245, 381 o-3820. Kawamura, M., Keim, P. S., Goto, Y., Zalkin, H., and Heinrikson, (1978), J. Biol. Chem., 253, 4659-4668. Grove, T. H., and Levy, H. R.T976), Biochim. Biophys. Acta., 464-474.
Chem.,
Service. REFERENCES
1. 2. 3.
to
mM is
the substrate
PRT by binding
PRPP or Mg"-PRPP.
with
residues
essential
and/or
between
that
substrate
on the AS-PRT enzyme complex
of the essential
amino
combination
the metal
bound per mole of enzyme even at a 70% level
identity
respect
A Ki of 0.44
suggests
have shown that
PLP apparently
additional
are
with
from
Kd = 12 UM (22).
In summary,
with
calculated
PLP is competitive
to anthranilate.
PRT is Mgf2 -PRPP and not PRPP alone,
PRPP, with
A Ki of 1.17 mM is
data.
The experiments PRPP afforded
kinetics.
s,tudies
PRPP and non-competitive
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
393
R. L. 5,
Vol.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
86,
No.
2, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Henderson, E. J . 3 Nagano, H., Zalkin, H., and Hwang, L. H. (1970), J. Biol. Chem., 245, 1416-1423. Biochem. J., 147, 351-358. Chen, S., and Engel, P. (1975), Levy, H. M., Leber. P. D.. and Ryan, E. M. (1963), J. Biol. Chem., "-238, 3654-3659. " Kitz, R., and Wilson, I. (1962), J. Biol. Chem., 237, 3245-3249. Anderson, B. M., Anderson, C. II., and Churchich, J. E. (1966), Biochemistry, 5, 2893-2900. Yost, F., and Harrison, J. (1971), Biochem. Biophys. Res. Commun., 42, 516-522. Rippa, M., and Pontremoli, S. (1969), Arch. Biochem. Biophys., 133, 112-118. R. B., Nolan, C., and Fischer, E. H. (1971), Forrey, A. W., Olsgaard, Biochimie., 3, 269-281. Chemical Modification of Means, G. E., and Feeney, R. E. (1971), Proteins, p. 134, Holden-Day, Inc., San Francisco. Colombo, G., and Marcus, F. (1974), Biochemistry, 13, 3085-3091. S., and Folkers, K. (1948), J. Am. Chem. Sot., 7D-, Heyl, D., Harris, 3429-3431. Buell, M. V., and Hansen, R. E. (1960), J. Am. Chem. Sot., 82, 6042-6049. J. Am. Chem. Sot., 79-, 485-490. Metzler, D. E. ,(1957), Heinert, D. and Martell, A. E. (1963), 3. Am. Chem. Sot., 85, 183-188. Morino, Y. and Snell, E. E. (1967), J. Biol. Chem., 242, 2800-2809. Milhausen, M. and Levy, H. R. (1975), Eur. J. Biochem., 3, 453-461. Hoppe-Seyler Z. Physiol. Chem., Domschke, W., and Domagk, G. F. (1969), 350, 1111-1116. Schnackerz, K. D., and Noltmann, E. A. (1971), Biochemistry, lo, 4837-4843. Robison, P. D., Nowak, T., and Levy, H. R. Arch. Biochem. Biophys. (in press). Henderson, E. J. and Zalkin, H. (1971), J. Biol. Chem., 246, 6891-6898.
394
