From the
The potential photoaffinity probe 8-azido-adenosine
(8-N
S-Adenosylhomocysteine (AdoHcy)
In contrast to the
coenzyme-binding site, little is known about the substrate-binding site
of AdoHcy hydrolase. Efforts have been made to elucidate information
about the active site residues that participate in catalysis or
substrate binding using strategies including chemical modification,
site-directed mutagenesis, and limited proteolysis. Two cysteine
residues, 1 arginine residue, one carboxyl group, and 1 histidine
residue were indicated to be essential for enzyme activity of the rat
liver AdoHcy hydrolase by chemical modification
studies(15, 16, 17, 18, 19) .
Site-directed mutagenesis studies showed that Lys
Another
potential approach to elucidating crucial amino acids at the active
site of an enzyme is photoaffinity labeling. Photoaffinity probes have
been used very effectively for studying nucleotide/nucleoside-binding
proteins(23, 24) . In earlier studies, Aiyar and
Hershfield (25) showed that 8-N
The photoaffinity probe 8-N
Since the earlier work by Hershfield et
al. (25) was done using AdoHcy hydrolase isolated from
human placenta while our studies were done with the recombinant human
placental enzyme, we first conducted experiments to confirm the
specificity of photolabeling of AdoHcy hydrolase with
8-N
On-line formulae not verified for accuracy where PA
When
the non-absorbed radioactive fractions in Fig. 4a were
pooled and analyzed by reverse phase HPLC, the chromatograph showed two
major radioactive peaks: peak a (fraction number 40-43)
and peak b (fraction number 44-46) (Fig. 5). These
two peaks account for about 70% of the total radioactivity. These two
peaks were pooled separately and rechromatographed by reverse phase
HPLC by manually collecting eluted peaks. Peaks associated with
significant radioactivity were rechromatographed. As shown in Fig. 6, rechromatography generated a major and several minor
components with the radioactivity associated only with the major
components, peak a` and peak b`, which were isolated
from peaks a and b, respectively.
Comparison of amino acid sequences of AdoHcy
hydrolase from different sources has provided useful information about
the structure-function relationships of the enzyme and the identity of
essential amino acid residues involved in enzyme catalysis or
substrate/cofactor binding(5, 21) . AdoHcy hydrolase has
been cloned from a range of sources representing billions of years of
evolution. The alignment of entire sequences of AdoHcy hydrolase from
different sources has been published (5). Fig. 7shows only the
sequences in the two photolabeled regions. Both of the photolabeled
peptides are located in very conserved regions of the entire gene. For
example, with the exception of the first residue, Val
AdoHcy hydrolase (23.5 µg) was incubated
with 50 µM of
[2-
We acknowledge Dr. Michael Hershfield for providing us
with a sample of E. coli transformed with a plasmid encoding
for human placental AdoHcy hydrolase.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Ado) was shown to serve as a substrate for the
3`-oxidative activity of human S-adenosylhomocysteine (AdoHcy)
hydrolase (Aiyar, V. N., and Hershfield, M. S.(1985) Biochem. J. 232, 643-650). In this study, we have determined the
equilibrium binding properties of 8-N
-Ado with AdoHcy
hydrolase (NAD
form) and identified the specific amino
acid residues that are covalently modified. After irradiation of the
reaction mixture of [2-
H]8-N
-Ado and
AdoHcy hydrolase (NAD
form) and followed by tryptic
digestion, peptides specifically photolabeled by
[2-
H]3`-keto-8-N
-Ado were effectively
separated from peptides nonspecifically labeled with
[2-
H]8-N
-Ado using boronate affinity
chromatography. After purification by reverse phase high performance
liquid chromatography, two photolabeled peptides were isolated and
identified as Val
-Lys
and
Val
-Arg
, in which Ala
and
Ile
were associated with radioactivity. The specificity
of the photoaffinity labeling with
[2-
H]3`-keto-8-N
-Ado was demonstrated
by the observation that these photolabeled peptides were not isolated
when [2-
H]8-N
-Ado was incubated with
apo AdoHcy hydrolase and irradiated. The two photolabeled peptides are
assumed to be parts of the adenine-binding domain for substrates. They
are both within well conserved regions of AdoHcy hydrolases. The
peptide Val
-Lys
is located very close to
Cys
and Glu
-Ser
, both of
which were indicated to be located in the active site of the enzyme by
chemical modification and limited proteolysis methods. The peptide
Val
-Arg
is adjacent to Leu
,
which is proposed by a computer graphics model to interact with the
C-6-NH
group of Ado.
(
)hydrolase (EC 3.3.1.1) catalyzes the reversible
hydrolysis of AdoHcy to adenosine (Ado) and L-homocysteine
(Hcy)(1) . The mechanism of the enzyme reaction has been
elegantly elucidated by Palmer and Abeles(2) . AdoHcy hydrolase
has been cloned from a number of different sources, including Homo
sapiens(3) , Rattus species(4) , Plasmodium falciparum(5) , Rhodobacter capsulatus (6), Triticum aestivum(7) , Catharanthus
roseus (8), Petroselinum crispum(9) , Leishmania donovani(10) , Dictyostelium
discoideum(11) , and Caenorhabditis
elegans(12) . The amino acid sequences of the cloned AdoHcy
hydrolases have been deduced from their cDNA sequences. Comparison of
the amino acid sequences from these species shows a remarkable degree
of conservation ranging from 64% identity between human and Rhodobacter capsulatus(6) to 97% identity between
human and rat(3) . A highly conserved amino acid region from 213
to 244 in the recombinant rat liver enzyme has been postulated to be
part of the NAD
-binding site (4) based on the
fingerprint sequence of G-X-G-X-X-G, which is a
characteristic
-fold formed in NAD
- and
FAD-binding domains of proteins that bind the ADP moiety of the
dinucleotide cofactor(13) . Mutations of any of the 3 glycine
residues produce mutant proteins that have no catalytic activity and
contain no bound coenzyme(14) .
is
critical for the activity of the human enzyme and Cys
has
an important role in the structure of the rat liver
enzyme(20, 21) . More recently, a limited proteolysis
study demonstrated that Glu
-Ser
is located
in or near the active site of the enzyme(22) .
-Ado, a potential
photoaffinity labeling reagent, was a substrate for the 3`-oxidative
activity of AdoHcy hydrolase. In this report, we have determined the
equilibrium binding properties of 8-N
-Ado with AdoHcy
hydrolase (NAD
form) and identified the specific amino
acid residues that are covalently modified after irradiation of the
substrate-enzyme complex.
Materials
8-N-Ado,
NAD
, NADH, and L1-tosyl-amido-2-phenylethyl
chloromethyl ketone-treated trypsin (Type XIII) were purchased from
Sigma. [2-
H]8-N
-Ado (17.7 Ci/mmol)
was obtained from Moravek Biochemicals Inc. (Brea, CA). Boronate
affinity resin Affi-Gel 601 was from Bio-Rad.
Purification of Recombinant AdoHcy
Hydrolase
Expression of the human placental AdoHcy hydrolase
cDNA in Escherichia coli and purification of the enzyme were
carried out as described previously(26) . The purified,
homogeneous enzyme is a tetramer consisting of four identical subunits
of M 47,000. In this study, the subunit M
was used to calculate the molarity of enzyme
solutions. The protein concentration was determined by the method of
Bradford (27) using bovine serum albumin as a standard.
Preparation of Apo AdoHcy Hydrolase
Apo AdoHcy
hydrolase was obtained by treatment of AdoHcy hydrolase (NAD form) with acidic (NH
)
SO
to
remove the NAD
as described
previously(28, 29) .
Assay of AdoHcy Hydrolase Activity
The AdoHcy
hydrolase activity was determined in the synthetic direction as
described earlier (26). This assay measures the rate of formation of
AdoHcy from Ado and Hcy using HPLC.
Determination of E
AdoHcy hydrolase (100 µg) was incubated with or
without the photoaffinity probe 8-NNAD
and
E
NADH
-Ado (50
µM) in 200 µl of 50 mM sodium phosphate
buffer, pH 7.2, (buffer A) for 1 h at 37 °C. To the reaction
mixture was added 3 volumes of 97% ethanol to denature the enzyme. The
precipitate was removed by centrifugation, and the supernatant was
lyophilized. The residue was then dissolved in 100 µl of water for
HPLC analysis using a reverse phase column (Econosphere, C18, 5 µm,
250
4.6 mm, Alltech, Deerfield, IL). NAD
and
NADH were eluted from the column isocratically with 0.1 M sodium phosphate buffer, pH 7.0, containing 2.5% of methanol at a
flow rate of 1.0 ml/min. Standard curves of NAD
and
NADH were constructed by using known concentrations of freshly prepared
authentic NAD
and NADH. The peaks were monitored at
260 nm.
Photoaffinity Labeling
AdoHcy hydrolase (23.5
µg) was incubated with
[2-H]8-N
-Ado (50 µM, 50
µCi/µmol) in the presence or absence of Ado in 50 µl of
buffer A at 37 °C for 1 h. The reaction mixture was chilled to 0
°C and loaded onto a piece of glass plate, which was placed on an
ice bath. The photolysis was performed by irradiating the sample with a
minelight hand lamp (UVGL-25, 1400 µW/cm
) at 254 nm
from a height of 7.6 cm for 2 min. Total binding was determined by a
gel filtration method using a Sephadex G-50 spin column. The column (3
ml) equilibrated with buffer A was prespun at 400
g for 5 min
using a swing rotor. The photolyzed protein sample was diluted to 300
µl with buffer A and loaded onto the prespun column. The column was
then spun for another 5 min at the same speed. The eluate was collected
in a test tube which holds the spin column during centrifugation. The
protein concentration of the eluate was determined by the Bradford
method and the radioactivity was determined by liquid scintillation
counting. The amount of covalent binding was determined by denaturing
the protein before gel filtration. The photolyzed sample was mixed with
250 µl of 10 M urea containing 10 mM
dithiothreitol, and heated at 100 °C for 3 min. After cooling to
room temperature, the sample was loaded into a prespun Sephadex G-50
column equilibrated with 1 M urea in buffer A. The column was
then spun for 5 min at the same speed as that for the prespin. The
protein concentration and radioactivity in the eluate were determined
as described above. Control experiments were performed under the
denatured conditions with the probe alone or with a non-irradiated
enzyme-probe mixture. In both cases, there was no detectable
radioactivity in the eluate.
Isolation of Photolabeled Peptides
AdoHcy
hydrolase (2 mg) was incubated with
[2-H]8-N
-Ado (50 µCi/µmol) in
a protein-to-probe molar ratio of 1:5 at 37 °C for 1.5 h. After
photolysis at 0 °C for 2 min, the photolabeled protein was filtered
through a Sephadex G-50 spin column equilibrated with 100 mM NH
HCO
, pH 8.4, (buffer B) (a 3-ml column
for each 300 µl of protein sample) as described above. The eluted
protein was denatured by adding solid urea to a final concentration of
8 M. The denatured protein was dialyzed against buffer B
containing 2 M urea for 16 h with 2 buffer changes followed by
another 6 h of dialysis against buffer B without urea. The dialyzed
protein was digested with trypsin in an enzyme-to-substrate ratio (w/w)
of 1:20 at 37 °C for 14 h. This was followed by addition of another
5% of trypsin for further digestion for 4 h.
Boronate Affinity Chromatography
The boronate
affinity chromatography was performed essentially as described by Haley et al.(30) . The digested protein was diluted
with 100 mM ammonium acetate, pH 8.9 (buffer C), and loaded
onto an Affi-Gel 601 column (3 ml) equilibrated with buffer C. The
column was washed with 20 ml of buffer C, 10 ml of buffer C containing
0.5 M NaCl, 10 ml of buffer C containing 4 M urea, 20
ml of buffer C containing 100 mM sorbitol, and finally 10 ml
of 0.1 M acetic acid. Fractions of 1 ml were collected.
Radioactivity in each fraction was determined by liquid scintillation
counting, and peptide concentration was monitored at 220 nm with
appropriate elution solutions as references. The non-absorbed
radioactive fractions were loaded onto another Affi-Gel 601 column to
ensure that the radioactive peptides in these fractions were not due to
overloading of the column. The pooled, non-absorbed radioactive
fractions were then concentrated by lyophilization.
Reverse Phase HPLC
The photolabeled peptides
collected from the non-absorbed radioactive fractions from the boronate
affinity column were purified by reverse phase HPLC on a Vydac C18
Protein and Peptide column (Vydac 218 TP54, C18, 5 µ, 250
4.6 mm). The solvent system consisted of mobile phase I (0.1%
trifluoroacetic acid) and mobile phase II (80% CH
CN/20%
H
O/0.07% trifluoroacetic acid). The elution was carried out
with 2% of mobile phase II in mobile phase I with a linear gradient to
70% mobile phase II over 120 min at a flow rate of 0.5 ml/min. The UV
absorbance of the eluted peptides was monitored at 220 nm. The
radioactivity in the fractions collected (0.5 ml) was measured by
liquid scintillation counting. Peptide peaks containing major
radioactivity were collected, concentrated by speed vacuum, and
rechromatographed on the same column using elution conditions of 20% of
mobile phase II with a linear gradient to 60% mobile phase II over 60
min. Peaks were collected manually and radioactivity was determined.
Radioactive peaks were rechromatographed again with the same elution
conditions as described above.
Identification of Labeled Peptides and Amino Acid
Residues
The manually collected peptide peaks containing
radioactivity significantly above background were sequenced by
automated Edman degradation on an Applied Biosystem 473A Protein
Sequencer at the Kansas State University Biotechnology Laboratory,
Manhattan, KS. At each sequencing cycle, the washing from the
conversion flask and eluate from the HPLC were collected for
determination of radioactivity.
-Ado was used
previously to study the relationship between the Ado and cyclic
AMP-binding sites on AdoHcy hydrolase purified from human
placenta(25) . It was shown that 8-N
-Ado was a
substrate for the the first step (oxidation of the 3`-hydroxyl group)
in the catalytic mechanism of AdoHcy hydrolase, resulting in conversion
of E
NAD
to E
NADH and
formation of 3`-keto-8-N
-Ado bound to the active site of
the enzyme. Based on this information, we felt that advantage could be
taken of the substrate activity of 8-N
-Ado to differentiate
specifically photolabeled peptides from nonspecifically photolabeled
peptides, a problem that often causes difficulties in photoaffinity
labeling experiments.
-Ado. To show the specificity of the photaffinity probe,
the photolabeling should be saturable and the photolabeling protected
by a natural substrate of the enzyme. As shown in Fig. 1, when
the recombinant human placental AdoHcy hydrolase was photolabeled with
increasing concentrations of
[2-
H]8-N
-Ado, the extent of labeling
approached saturation at approximately 40 µM. When the
enzyme was incubated with 50 µM
[2-
H]8-N
-Ado in the presence of
increasing concentrations of Ado, a natural substrate of the enzyme,
but without irradiation, 95% of the total binding of the photolabeling
probe was inhibited at 200 µM Ado (Fig. 2). This
result indicated that 8-N
-Ado and Ado bind to the same site
on AdoHcy hydrolase. An apparent dissociation constant (K
) of 6.8 µM for
8-N
-Ado was calculated using a computer-aided curve fitting
with the following equation:
was the protein-photolabeling probe complex
formed in the presence of Ado; PA was the complex formed in the absence
of Ado; A was the concentration of 8-N
-Ado; and K
` was the dissociation constant (4.5
µM) of Ado determined separately by equilibrium dialysis.
However, upon irradiation of the enzyme with
[2-
H]8-N
-Ado in the presence of Ado,
the protection was only 60% at 200 µM Ado, and further
increases of Ado concentration did not afford better protection (Fig. 2). This suggested that not all photolabeling occurred in
the Ado-binding site and that some of the 8-N
-Ado must have
nonspecifically photolabeled the protein outside of the Ado-binding
site, which could not be protected by Ado. summarizes the
results of binding, photoincorporation, and inactivation of AdoHcy
hydrolase by 8-N
-Ado. Incorporation of
[2-
H]8-N
-Ado to the protein was
light-dependent. Incubation of the enzyme with the photolabeling probe
in the absence of light gave a binding stoichiometery of 0.81 ±
0.05 mol of probe/mol enzyme subunit, but without covalent
incorporation. After irradiation, the total binding increased to 0.95
± 0.06 mol of probe/mol of enzyme subunit, in which 0.29
± 0.05 mol of the probe was covalently photoinserted into the
protein. However, about 40% of the covalent modification could not be
protected by high concentrations of Ado, indicating the existence of
significant nonspecific photoinsertion. This result was in agreement
with the earlier observation that 20-60% of the covalent labeling
of AdoHcy hydrolase with 8-N
-Ado could not be blocked by
Ado (25). Subtracting the nonspecific labeling from the total
photoinsertion resulted in a net amount of specific photoincorporation
of 18%, which is comparable to the reported value of
5-14%(25) . The photoincorporation and specificity varied
with the enzyme probe ratio. An enzyme/probe ratio of 1:4-6 was
found to give the highest photoincorporation with the lowest
nonspecific labeling under the conditions of 2 min of irradiation.
Irradiation caused a time-dependent loss of the enzyme activity. About
15% of the enzyme activity was lost in the 2-min irradiation in the
absence of the probe, and 56% was lost in the presence of the probe.
This increased loss of the enzyme activity was prevented by inclusion
of 200 µM Ado (). 8-N
-Ado in the
absence of irradiation was a slow time-dependent inhibitor of AdoHcy
hydrolase. Incubation of the enzyme with 50 µM of
8-N
-Ado at 37 °C for 1 h in the absence of light
resulted in 51% inactivation of the enzyme. This enzyme inactivation
could be protected by Ado, supporting the idea that the probe was
interacting at the active site of the enzyme.
Figure 1:
Saturation of photoinsertion into
AdoHcy hydrolase by [2-H]8-N
-Ado.
Recombinant human placental AdoHcy hydrolase (23.5 mg) was incubated
with increasing concentrations of
[2-
H]8-N
-Ado (50 µCi/µmol)
for 1 h at 37 °C in 50 µl of 50 mM sodium phosphate
buffer, pH 7.2 (buffer A), and irradiated for 2 min at 0 °C. The
photolabeled protein was denatured and chromatographed on a Sephadex
G-50 spin-column under denatured conditions as described under
``Experimental Procedures.'' Radioactivity in the eluate from
the spin column was determined by liquid scintillation
counting.
Figure 2:
Effect of Ado on binding and
photoinsertion of [2-H]8-N
-Ado into
AdoHcy hydrolase. Recombinant human placental AdoHcy hydrolase (23.5
µg) was incubated with 50 µM of
[2-
H]8-N
-Ado (50 µCi/µmol) in
the absence and presence of increasing concentrations of Ado in 50
µl of buffer A at 37 °C for 1 h. Non-irradiated samples (
)
were used to determine the total binding, and irradiated samples
(
) were used to determine the covalent binding as described
under ``Experimental Procedures.'' The ratio of the binding
stoichiometry (PA/protein-photolabeling probe complex) in the presence
of Ado to that in the absence of Ado was plotted against Ado
concentration.
Because of the high
percentage of nonspecific photoinsertion, one might, at first glance,
question the strategy of identifying specifically photolabeled peptides
of AdoHcy hydrolase using 8-N-Ado as a probe. However, if
one takes advantage of 8-N
-Ado being a substrate of the
oxidative activity of the enzyme(25) , it immediately becomes
apparent that it should be possible to separate the peptides
specifically labeled with 3`-keto-8-N
-Ado from those
nonspecifically labeled by 8-N
-Ado by using boronate
affinity chromatography. Boronate resin has a strong affinity for
compounds that have adjacent cis-hydroxyl group(s) (cis-diols) and has thus been used to separate
deoxyribonucleotides from ribonucleotides effectively(31) .
8-N
-Ado bound at the enzyme active site will participate in
the enzyme-catalyzed oxidation reaction, which reduces E
NAD
to E
NADH with the
concomitant formation of 3`-keto-8-N
-Ado(25) . The
NADH form of the enzyme, in turn, tightly binds the 3`-keto product in
its active site(32) , which becomes covalently bound after
irradiation. Peptides labeled with 3`-keto probe are thus assumed to be
parts of the adenine (Ade)-binding domain. Because the 3`-keto probe
lacks adjacent cis-hydroxyl groups, peptides labeled by
3`-keto-8-N
-Ado should have no affinity to the boronate
resin and should be eluted from the column directly without retardation
(non-absorbed fractions). In contrast, peptides nonspecifically
modified by 8-N
-Ado should possess adjacent cis-hydroxyl groups (2` and 3`- hydroxyl group) and hence have
a strong affinity to the boronate resin and remain in the column. In
order to utilize this affinity separation technique, we first undertook
an experiment to confirm that 8-N
-Ado serves as a substrate
for the oxidative activity of the enzyme by monitoring the conversion
of E
NAD
to E
NADH after
incubation of the enzyme with the probe. As shown in Fig. 3, the
purified AdoHcy hydrolase contains about 0.85 mol of NAD
and 0.1 mol of NADH/ mol of enzyme subunit (Fig. 3a). After incubation with 50 µM of
8-N
-Ado at 37 °C for 1 h, about 53% of the
NAD
was converted to NADH (Fig. 3b).
Prolonged incubation did not significantly increase the ratio of
NAD
/NADH. This result confirmed the earlier
observation that 8-N
-Ado participates as a substrate in the
enzyme-catalyzed oxidation reaction.
Figure 3:
8-N-Ado induced conversion of
AdoHcy hydrolase-bound NAD
to NADH. Recombinant human
placental AdoHcy hydrolase (100 µg) was incubated with
8-N
-Ado (50 µM) in 200 µl of buffer A for
1 h at 37 °C. The protein was denatured by mixing with 3 volumes of
ethanol, and the nucleotides released were analyzed on a reverse phase
HPLC column (Econosphere, C18, 5 µ, 250
4.6 mm) using a
neutral mobile phase as described under ``Experimental
Procedures.'' a, enzyme alone; b, enzyme
incubated with 8-N
-Ado; c, authentic
NAD
and NADH.
After irradiation of the
reaction mixture of AdoHcy hydrolase and 8-N-Ado, the
protein was denatured by treatment with 8 M urea and
subsequent heating. The free probe was removed by gel filtration and
dialysis. The labeled protein was digested with trypsin, and the
resulting peptides were subjected to boronate affinity chromatography. Fig. 4a shows a typical elution profile of the
photolabeled tryptic peptides on boronate affinity chromatography. Two
major radioactive peaks were observed; one eluted with the non-absorbed
fractions (fractions 2-7) and accounted for about 40% of the
total radioactivity, and the second was eluted by 100 mM sorbitol (fractions 44-56). To confirm that the radioactive
peptides in the non-absorbed fractions were not due to overloading the
column, those fractions were pooled and applied to another boronate
column. From this chromatography, more than 95% of the radioactivity
was recovered from the non-absorbed fractions (data not shown). This
result indicates that the non-absorbed radioactive peptides contain no
adjacent cis-hydroxyl group(s) and were more than likely
labeled by 3`-keto-8-N
-Ado. The percent (40%) of
specifically photolabeled peptides from the affinity column was lower
than that (60%) observed from the Ado protection experiment, indicating
that some of the active site peptides were labeled by
8-N
-Ado rather than its 3`-keto derivative. This was
expected since incubation of the enzyme with 8-N
-Ado at 37
°C for 1 h could convert only 53% of the E
NAD
to E
NADH, and the
rest of the probe bound in the active site still existed in its
original non-oxidized form at the time point of irradiation.
Nevertheless, this part of the specifically labeled peptides was
sacrificed in the separation method.
Figure 4:
Boronate affinity chromatography of
photolabeled tryptic peptides of AdoHcy hydrolase. Two mg of
recombinant human placental AdoHcy hydrolase (NAD form) or apo form was photolabeled with
[2-
H]8-N
-Ado (50 µCi/µmol)
and digested with trypsin as described under ``Experimental
Procedures.'' The digest was loaded onto a 3-ml Affi-Gel 601
equilibrated with 100 mM ammonium acetate buffer, pH 8.9
(buffer C), and eluted with various solutions as indicated in the
figure. Fractions of 1 ml were collected. Radioactivity (
,
)
in each fraction was determined by liquid scintillation counting, and
peptide concentration (broken line) was monitored at 220 nm. a, NAD
form of AdoHcy hydrolase; b,
apo form of AdoHcy hydrolase.
To further confirm the
specificity of the photoinsertion by 8-N-Ado, a parallel
photolabeling experiment was carried out using apoAdoHcy hydrolase
which lacks NAD
and thus does not catalyze
3`-oxidation of substrates. However, apoAdoHcy hydrolase still binds
Ado and catalyzes the 5`-hydrolytic reaction (28). As shown in Fig. 4b, approximately 95% of the total radioactivity
from the 8-N
-Ado-apoAdoHcy hydrolase experiment was
retained on the boronate affinity resin and was eluted specifically by
sorbitol. The small amount (5%) of the radioactivity that elutes with
the non-absorbed fractions most likely arises from the residual
NAD
form of the enzyme present in the apo enzyme.
Comparison of the elution profiles of boronate affinity
chromatographies of the NAD
form and the apo form of
the enzyme photolabeled with
[
H]8-N
-Ado gives strong evidence that
radioactive peptides in the non-absorbed fractions from the affinity
column are those specifically photolabeled by enzymatically oxidized
8-N
-Ado, presumably 3`-keto-8-N
-Ado.
Figure 5:
Reverse phase chromatography of
photolabeled peptides of AdoHcy hydrolase. Fractions containing
photolabeled peptides from Fig. 4a (fractions 2-7) were
pooled, concentrated, and applied to a Vydac C18 Protein and Peptide
column (Vydac 218TP54, C18, 5 µ, 250 4.6 mm). Elution was
carried out by a linear gradient of mobile phase II
(acetonitrile/H
O, 0.07%trifluoroacetic acid) in mobile
phase I (0.1% trifluoroacetic acid) from 2 to 70% over 120 min at a
flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected, and
radioactivity in each fraction was measured by liquid scintillation
counting.
Figure 6:
Rechromatography of photolabeled peptides
purified from HPLC. Peak a (fraction 40-43) and peak b (fraction 44-46) in Fig. 5 were pooled, concentrated,
and loaded onto a reverse phase HPLC column (Vydac 218TP54, C18, 5
µ, 250 4.6 mm). Peptides were eluted by a gradient of
mobile phase II (acetonitrile/H
O, 0.07%trifluoroacetic
acid) in mobile phase I (0.1% trifluoroacetic acid) from 20 to 60% over
60 min at a flow rate of 0.5 ml/min. Peaks were collected manually, and
radioactivity was determined for each peak. The major radioactive peaks
were loaded onto the same column again and eluted under the same
conditions. Shadowed peaks were radioactive and were manually
collected for amino acid sequencing.
Upon amino acid
sequence analysis, peak a` was found to contain peptide
Val-Arg
(peptide I), and peak b`
contained peptide Val
-Lys
(peptide II) as
seen in . Liquid scintillation counting of the washing
from the conversion flask and the eluate from HPLC at each cycle of
sequencing showed that Ile
in peptide I and Ala
in peptide II were associated with the highest radioactivity in
the two peptides. These results indicate that Ile
and
Ala
are the photoinsertion sites. Since both Ile
and Ala
were eluted with normal retention times
from the reverse phase HPLC during sequencing, it is likely that the
H label dissociated from the modified amino acid residue
during the sequencing. In a separate experiment, the photolabeled
peptides were found to be acid-labile. Upon exposure of the labeled
peptides to anhydrous acetic acid, the
H label completely
dissociated from peptides. Therefore, it is probable that during the
sequencing, the
H label dissociated in the conversion flask
where anilinothiozolinone amino acids are converted to
phenythiohydantoin derivatives at high temperature (
64 °C) and
low pH (<2.0).
,
the photolabeled region of peptide II is almost completely conserved in
all known AdoHcy hydrolases. The only exceptions are the P.
falciparum enzyme, where 3 residues are replaced, and the L.
donovani enzyme, where 2 residues are substituted. Nevertheless,
the photoinsertion site Ala
is conserved in all known
AdoHcy hydrolases. Interestingly, peptide II is located very close to
Cys
, a residue that was identified as being located in
the active site of the enzyme by chemical modification.
(
)Moreover, limited proteolysis studies on human AdoHcy
hydrolase from our laboratory have recently demonstrated that
Glu
-Ser
is located in or near the active
site of the enzyme by showing that cleavage of the peptide bond between
Glu
-Ser
by protease Staphylococcus
aureus strain V8 on a mutant form (K426E) of the enzyme was
specifically protected by the substrate Ado(22) . Information
from these two separate studies gives further support to the idea that
peptide II could be located in or near the active or substrate-binding
site of the enzyme. In fact, Ala
may be located very
close to the C-8 position of Ade in the three-dimensional structure of
the enzyme, at least after conformational changes induced by the probe
(32). For the photolabeled peptide I
(Val
-Arg
), a region from the third residue
(Ile
) of the peptide to Arg
is well
conserved with only a few substitutions observed in enzymes from lower
evolved cells such as parasites and bacteria. In fact, this region was
predicted to be involved in Ade ring binding by a computer graphics
model developed in our laboratory, which shows that in the Ade portion
of substrates and inhibitors, the C-6-NH
group interacts
with the main-chain carbonyl group of Leu
(Leu
in human) for recombinant rat liver enzyme(33) . Since
Leu
in the human enzyme is only 9 residues away from the
photoinsertion site Ile
, it is likely that Ile
is positioned such that it is close enough to the C-8 of the Ade
ring to which (C-8) the azido group is attached in the photoaffinity
probe. Therefore, based on results from this and earlier studies, we
believe that the two photolabeled peptides
Val
-Lys
and Val
-Arg
could be parts of the Ade ring-binding domain in the substrate
binding site of AdoHcy hydrolase.
Figure 7:
Amino acid sequence comparison of
photolabeled regions of AdoHcy hydrolases. The underlined regions indicate
[2-H]8-N
-Ado-photolabeled peptides,
and the boxed regions indicate residues that have been
proposed to have roles in catalysis or binding. Sequences: a, P. falciparum (5); b, R. capsulatus (6); c, T. aestivum (7); d, C. roseus (8); e, P. crispum (9); f, L.
donovani (10); g, D. discoideum (11); h, C. elegans (12); i, Rattus species (4); and j, Homo sapiens (3). Numbering is that
of the human enzyme sequence starting from the initial methionine
(3).
Table: Binding,
photoincorporation, and inactivation of AdoHcy hydrolase by
8-N-Ado
H]8-N
-Ado in 50 µl of buffer A
at 37 °C for 1 h in the presence and absence of Ado followed by
irradiation at 0 °C for 2 min. The remaining enzyme activity was
determined in the synthetic direction as described under
``Experimental Procedures.'' Stoichiometries of the total
binding were determined by gel filtration under non-denatured
conditions, and the covalent binding was determined by treating samples
with 8 M urea containing 10 mM dithiothreitol and
heating at 100 °C for 3 min before gel filtration under denatured
conditions as described under ``Experimental Procedures.''
Table: Amino acid
sequence analysis of the photolabeled peptides
-Ado,
8-azido-adenosine; HPLC, high performance liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.