(Received for publication, May 23, 1995; and in revised form, June 30, 1995)
From the
The enzyme 5-phosphoribosyl--1-pyrophosphate (PRPP)
synthetase from Escherichia coli was irreversibly inactivated
on exposure to the affinity analog 2`,3`-dialdehyde ATP (oATP). The
reaction displayed complex saturation kinetics with respect to oATP
with an apparent K
of approximately 0.8
mM. Reaction with radioactive oATP demonstrated that complete
inactivation of the enzyme corresponded to reaction at two or more
sites with limiting stoichiometries of approximately 0.7 and 1.3 mol of
oATP incorporated/mol of PRPP synthetase subunit. oATP served as a
substrate in the presence of ribose-5-phosphate, and the enzyme could
be protected against inactivation by ADP or ATP. Isolation of
radioactive peptides from the enzyme modified with radioactive oATP,
followed by automated Edman sequencing allowed identification of
Lys
, Lys
, and Lys
as probable
sites of reaction with the analog. Cysteine 229 may also be labeled by
oATP. Of these four residues, Lys
is completely conserved
within the family of PRPP synthetases, and Lys
is found
at a position in the sequence where the cognate amino acid
(Asp
) in human isozyme I PRPP synthetase has been
previously implicated in the regulation of enzymatic activity. These
results imply a functional role for at least two of the identified
amino acid residues.
5-Phosphoribosyl--1-pyrophosphate (PRPP) (
)synthetase (ATP: D-ribose-5-phosphate
pyrophosphotransferase EC 2.7.6.1)is a paradigm of the small subclass
of nucleotide-utilizing enzymes catalyzing reactions involving
nucleophilic attack at the
-phosphoryl group of the nucleoside
triphosphate chain(1, 2, 3) . The product
PRPP is an important precursor in several major biosynthetic pathways,
being utilized in de novo nucleotide biosynthesis, nucleobase
salvage, pyridine coenzyme production, and in plants and
microorganisms, the biosynthesis of tryptophan and
histidine(4, 30) . Thus, a detailed understanding of
the mechanism of catalysis and the regulation of this enzyme is
important from both an enzymological and physiological point of view:
The enzyme serves as a ``mechanistic bridge'' between the far
more common enzymes that catalyze nucleophilic attack at the
- and
-phosphates (e.g. nucleotidyltransferases and kinases,
respectively), and PRPP is an important metabolite, and thus a
potential control point, in intermediary metabolism.
The reaction
catalyzed by PRPP synthetase is ATP + Rib-5-P PRPP +
AMP. The enzyme requires the presence of divalent metal ions for
activity, with the preferred cation being
Mg
(5, 6) . Furthermore, Escherichia coli and Salmonella typhimurium PRPP
synthetases are inhibited by ADP through binding at the active site and
at an allosteric site specific for this
nucleotide(7, 8, 9) . In spite of the fact
that PRPP synthetases from several diverse species have been cloned and
sequenced (e.g.(10, 11, 12, 13, 14) ),
and that some of these have been purified to homogeneity and
characterized(9, 15, 16, 17, 18) ,
there exists surprisingly little information about the nature of
individual amino acids present in the active site of PRPP synthetase
and how they participate in catalysis or substrate binding. Even less
is known about the types of amino acid residues present in the
allosteric site and their role in the control of enzymatic activity. In
the absence of a high resolution three-dimensional structure,
identification of functionally important residues has been limited to
indirect methods such as chemical modification, mutagenesis, genetic
methods, and comparative sequence analysis. Affinity labeling studies
with 5`-p-fluorosulfonylbenzoyladenosine(19) ,
mutagenesis studies(20) , and characterization of mutants
isolated by genetic methods (21) have led to the identification
of a region of highly conserved amino acid sequence within the PRPP
synthetase family. This region may be involved in ATP or divalent
cation binding and includes a histidyl residue that may act as a
general base during catalysis. Sequence comparisons with other proteins
in the data base(9) , in combination with mutagenesis
studies(22, 23) , have identified another conserved
region possessing similarity to the phosphoribosyltransferases and
containing conserved aspartyl residues which may be involved in Rib-5-P
or PRPP binding.
Chemical modification studies using the group-specific reagent pyridoxal-5-phosphate implicated one or more lysyl residues as being functionally important in S. typhimurium PRPP synthetase. Pyridoxal-5-phosphate was postulated to react at the active site of the enzyme near the Rib-5-P-binding site(24) . The affinity analog prepared by treating ATP with sodium periodate, oATP(25) , presents an attractive opportunity to identify this putative functional lysyl residue because the reagent contains the same reactive moiety as pyridoxal-5-phosphate and should be directed to the active site of the enzyme by virtue of its similarity to the natural substrate ATP. The reagent has previously been successfully used to identify lysyl residues in the active sites of several enzymes(26, 27, 28) . Furthermore, it is easily prepared, and any nucleotide with free 2`,3`-hydroxyls can be prepared in the same manner. This will allow use of the periodate derivative of the allosteric effector ADP to probe the allosteric site of PRPP synthetase.
We report here the identification of active-site residues through affinity labeling studies utilizing oATP and E. coli PRPP synthetase.
Figure 1:
Kinetics and stoichiometry of the
reaction of oATP with PRPP synthetase. Reaction conditions were as
described under ``Experimental Procedures.'' At defined time
points, samples were removed from the reaction mix and subjected to the
spun column technique as described under ``Experimental
Procedures.'' The resulting eluate fractions were assayed for
enzymatic activity, protein concentration and, in stoichiometric
determinations, radioactivity. A, the time course of the
reaction in the presence of 2 mM oATP. The inset shows the double-reciprocal plot of the pseudo-first-order rate
constants for the reaction of PRPP synthetase with varying
concentrations of oATP. The pseudo-first-order rate constant, k, was calculated as an average value consisting
of the rate constants for the fast reaction and the slow reactions. The arrow indicates the value at which the apparent K
was determined. B, the
stoichiometry of inactivation of PRPP synthetase with
[2,8-
H]oATP.
The stoichiometry of the inactivation of PRPP
synthetase was determined using [8-C]oATP or
[2,8-
H]oATP. Fig. 1B shows the
relation between inactivation of the enzyme and incorporation of the
analog. Again, the biphasic form of the curve suggests two or more
heterogeneous reaction sites. Extrapolation of the data to complete
inactivation for the first site of reaction yielded a value of about
0.7 mol of oATP incorporated/mol subunit of the enzyme. Extrapolation
of the second site of reaction yields a value of about 1.3 mol of oATP
incorporated/mol subunit. The stoichiometry of inactivation was
somewhat variable and ranged from 0.5 to 0.8 for the first site and 1.1
to 1.5 for the second site.
The enzyme was protected from inactivation and incorporation of radioactive oATP when ATP was present in the reaction mix. Table 1shows the residual activity of PRPP synthetase after 3 h of reaction with oATP when varying concentrations of ATP were present in the mixture from the beginning. ADP at similar concentrations was also able to protect the enzyme against inactivation with oATP (data not shown). The latter agrees well with the fact that ADP can function as a competitive inhibitor with respect to ATP(7, 8) .
To bolster the argument that oATP binds and reacts at the active site, oATP was tested as a substrate for the PRPP synthetase reaction. oATP is altered in the ribose moiety of ATP, but the phosphate groups of oATP are probably intact. The enzyme activity was assayed using the coupled assay described under ``Experimental Procedures.'' PRPP was consistently formed in molar amounts that were equivalent to the utilization of about 50% of the added oATP when used as a pyrophosphoryl donor. The reaction was at least an order of magnitude slower than the reaction utilizing ATP as a substrate. No PRPP was formed in the absence of ATP or oATP. oATP has previously been shown to act as a substrate in several enzymatic reactions(26, 28, 35) .
The results of the kinetic, stoichiometric, and protection studies discussed above, combined with the fact that E. coli PRPP synthetase can use oATP as a substrate are compelling evidence that oATP is functioning as an ATP analog and reacting in a limited fashion at the active site of the enzyme. This is further supported by the proteolytic analyses of oATP-modified enzyme discussed below.
Figure 2:
Chromatographic analysis and isolation of
radioactive tryptic peptides from [H]oATP
modified PRPP synthetase. The modified enzyme was prepared by labeling
20 mg of PRPP synthetase at a concentration of 1 mg/ml with 2 mM [
H]oATP as described under
``Experimental Procedures.'' A, the UV elution
profile at 220 nm of a 500-µg sample of tryptic peptides from
[
H]oATP-labeled PRPP synthetase chromatographed
as described under ``Experimental Procedures.'' The gradient
profile is indicated and consists of an initial hold at 5%
CH
CN followed by a linear increase to 47.5%
CH
CN, then followed by a final increase to 90%
CH
CN with return thereafter to initial conditions.
Fractions of 400 µl were collected. For panels B and C, 50 µl of each 400-µl fraction was assayed for
radioactivity. The numbers in panel A correspond to
the peaks with associated radioactivity in panels B and C. B, (
): the profile of radioactivity obtained
from chromatography of tryptic peptides resulting from 4 h of treatment
of [
H]oATP-modified enzyme with TPCK-treated
trypsin; (
): the profile of radioactivity obtained from
chromatography of tryptic peptides resulting from PRPP synthetase
modified with [
H]oATP in the presence of 4
mM ATP. C, change in distribution of radioactivity
from chromatography of tryptic peptides isolated from a similar
preparation of [
H]oATP-modified PRPP synthetase
as described above, but which had been digested with TPCK-treated
trypsin overnight (
12 h) This preparation had a similar UV elution
profile to that shown in panel A.
To investigate whether the
appearance of these radioactive peaks was due to specific labeling by
oATP at the ATP-binding site, the enzyme was incubated with
[H]oATP under the same conditions as described
above, but in the presence of 4 mM ATP. The radioactivity
profile shown by the open circles in Fig. 2B clearly demonstrates that all sites are protected against reaction
with oATP when ATP is present. This strongly supports the results
obtained from the reaction kinetics, stoichiometry, and protection
studies demonstrating that oATP functions as an ATP affinity analog for
PRPP synthetase.
The pattern of radioactivity observed in Fig. 2B (closed circles) was somewhat
variable. The relative distribution of radioactivity between peaks 1
and 2 appears to depend on several factors. Fig. 2C shows the effect of extended TPCK-treated trypsin digestion
(12 h) on a similar preparation of
[
H]oATP-labeled enzyme. A new peak, peak 3, which
elutes at the column void volume is observed. In addition, a similar
peak is observed in chromatographic separations of peptides derived
from digestions with AspN and V8 proteases (data not shown). Peak 3 may
be a breakdown product of the oATP-enzyme adduct. This breakdown
product may be generated by chromatography of peptides derived from the
modified enzyme in the acidic trifluoroacetic acid/CH
CN
solvent system, or by extended incubation times at 37 °C during
proteolysis. That breakdown was the cause of the appearance of peak 3
is based upon four observations: (i) the radioactivity profile of pure
[
H]oATP chromatographed in this system yields a
peak that also elutes at the void volume; any breakdown product might
be expected to have similar chromatographic properties to authentic
oATP in this system; (ii) rechromatography of pure, undigested
[
H]oATP-labeled PRPP synthetase previously
isolated by chromatography in this system yields two peaks: the
undigested, labeled PRPP synthetase subunit eluting at 62 min, and a
peak eluting at the void volume; (iii) rechromatography of peaks 1 and
2 yields radioactive peaks eluting at the void volume; and (iv) amino
acid analysis indicates that the oATP-enzyme adduct is unstable when
exposed to acidic conditions. Alternatively, peak 3 may be a very
small, oATP-labeled peptide not retained on the C
column.
In the case of trypsin, this peptide could be generated from slow
cleavage at an oATP-modified lysyl residue. Thus, this peak would
appear only after extended treatments with trypsin. Breakdown of
oATP-peptide adducts has been previously
reported(26, 36) , while the results of our sequencing
studies discussed below suggest that slow tryptic cleavage at a
modified lysyl residue may occur. It is, however, more likely that
breakdown is the largest factor contributing to the appearance of peak
3 in tryptic digests of [
H]oATP-labeled PRPP
synthetase, given the results with other proteolytic digestions, and
with the undigested, labeled enzyme.
The isolated radioactive peptides contained sequences
that include lysines 181, 193, and 230 (see Fig. 3). The
sequence of peptide 2 overlapped the sequence of the peptide 1B. This
could be the result of slow cleavage at a modified lysyl residue
(Lys) as described in the previous section or,
alternatively, partial modification of Lys
. It is
important to note that, regardless of the peptide sequenced,
significant radioactivity was only found associated with the PTH
fraction from the first sequencing cycle of each peptide. There was
very little radioactivity retained on the sequencing filter and none
detected in the waste effluent from the sequencer. Thus, it is likely
that the radioactivity in the oATP-peptide adduct is lost under the
relatively harsh conditions encountered during the sequencing cycle.
The mechanism of this breakdown is not known, but several breakdown
species are possible. Radioactive isotope is present in the purine ring
of the analog, and the purine ring can be lost under acidic or basic
conditions. Furthermore, the chromatogram from the PTH analysis of
cycle 1 of each peptide revealed the presence of a UV absorbing peak
(
= 269 nm) eluting closely to PTH-Asp. This peak was
present in anomalously large amounts and was not present when peptides
isolated from unmodified PRPP synthetase were sequenced. These results
are consistent with the suggestion that this UV absorbing peak is
derived from the purine ring or adenosyl portion of the oATP-enzyme
adduct. Finally, trace amounts of radioactivity (3-5% of the
total radioactivity in the isolated peptide applied to the sequenator)
could be detected in the PTH fractions corresponding to the modified
lysines when very high specific activity oATP (5.5
10
disintegrations/min/µmol) was used to label PRPP synthetase.
Further evidence that these lysyl residues are the sites of
modification is provided by comparison of the yield of PTH lysine
derivatives present at suspected modification sites to the overall
yield of the other PTH-derivatives present in the sequence. The amounts
of the PTH-lysine at these sites (shown in boldface in Table 2)
were reduced 50-100% when compared to the expected yield based
upon the PTH-derivatives in the previous cycles. Recovery of the final
PTH-derivative at the end of peptides is often low, but a significant
increase in yield at this position is very unusual. In peptides 1B and
2, recovery of the the final arginines increase by 50 and 30%,
respectively. Thus, it is likely that this increase represents an
anomalously low yield in the preceding cycle, consistent with
assignment of the modification of Lys
at that position.
Firm assignment of Lys
as a site of labeling in peptide
1A was more problematic. The absence of an identifiable PTH-derivative
at the position corresponding to Cys
could be because of
poor recovery of peCys, or modification at this site. These residues
are well in from the N terminus of the peptide at a position where the
sequence signal begins to decay, and no readable sequence was detected
after Lys
in peptide 1A. Alternatively, cross-linking by
oATP of adjacent lysine and cysteine residues has previously been
postulated(36) .
Figure 3:
Alignment of PRPP synthetase amino acid
sequences in the region of oATP labeling. Numbers indicate the
amino acid from the N terminus of the polypeptide in the cases of the E. coli(9) , S.
typhimurium(10, 41) , B.
subtilis(13, 15) , and human (16, 42) enzymes. The other proteins are numbered from
the initiator methionines of the amino acid sequence deduced from the
respective DNA or cDNA sequence. The magenta lysyl residues in
the E. coli sequence indicate the sites of reaction with oATP.
The blue lysyl residues in the other sequences show lysyl
residues that may be equivalent to Lys in E. coli PRPP synthetase. The green residues in the E. coli sequence show the region identified as a putative PRPP-binding
site based upon homology to the phosphoribosyltransferases(9) . Asterisks above amino acids indicate identical residues among
the sequences. Segments 1-3 correspond to segments 1, 2,
and 3, respectively, of sequences suggested to be involved in ATP
binding in adenylate kinases and related proteins(14) . Vertical boxes indicate the amino acids in the other enzymes
that occupy homologous positions to the sites of labeling in the E.
coli enzyme. The radioactive peptides isolated in this work are
indicated by the labeled bars over the respective region of
amino acid sequence. The Leishmania donovani cDNA sequence has
been published(11) . The unpublished nucleotide sequences shown
were retrieved from the GenBank/EMBL Data Bank and have the following
accession numbers: Arabidopsis thaliana, X83764; Bacillus
caldolyticas, X83708; Listeria monocytogenes, M92842; Caenorhabditis elegans, U00036; Synechococcus sp.,
D14994.
To strengthen the argument that Lys is labeled by oATP, and to investigate whether Cys
was also labeled, isolation and sequencing of radioactive
peptides from digestion of [
H]oATP-labeled PRPP
synthetase with AspN protease was undertaken. Sequence analysis of a
radioactive AspN peptide containing Lys
is also shown in Table 2. Inspection of the PTH-derivative yields indicates that
the Lys
position indeed shows a decreased yield of
PTH-Lys, indicative of labeling at this position. While peCys was not
quantitated, inspection of the chromatogram of this cycle shows that
the yield of PTH-peCys is reduced only slightly (data not shown). This
result implies that modification of Cys
by oATP was
either very minor, or that the adduct of oATP with cysteine, presumably
a thiohemiacetal, is even more unstable to the sequencing conditions
than the lysine adduct. An interesting side note was that this AspN
peptide's C terminus was generated by cleavage before a glutamyl
residue in the PRPP synthetase sequence. The manufacturer's
product information sheet states that AspN protease cleaves at aspartyl
residues 2000 times faster than at glutamyl residues.
The
observation that these three lysyl residues would all be contained
within a single large peptide generated from treatment of modified
enzyme with S. aureus V8 protease under conditions where
cleavage is restricted to glutamic acid residues (37) prompted
us to examine V8 digests of modified enzyme by RP-HPLC. A single
radioactive peptide accounting for 80-90% of the amount of
radioactivity applied to the chromatograph was observed on RP-HPLC
analysis of V8 digested [H]oATP-modified PRPP
synthetase (data not shown). The rest of the radioactivity eluted at
the column void volume. This result in combination with the results
discussed above, supports our assignment of Lys
,
Lys
, Lys
as sites of reaction of the enzyme
with oATP.
Our affinity labeling results localize the sites of reaction of oATP with PRPP synthetase to a region of sequence spanning approximately 60 amino acid residues. Fig. 3shows an alignment of this sequence with the homologous amino acid sequences of known PRPP synthetases isolated from several diverse species.
Two of the
identified residues, Lys and Lys
are
especially noteworthy. Lysine 193 is completely conserved among all
known PRPP synthetases. Only two lysyl residues are completely
conserved in the PRPP synthetase family. Strong conservation of
Lys
across broad phylogenetic distances, in combination
with our results indicating that it lies in the active site, implies
that the residue possesses an important function in the protein.
Interestingly, this residue lies at the end of a stretch of sequence (segment 2, Fig. 3) that has similarity to one of three
sequence segments found in adenylate kinases and other enzymes (14, 38) . The segment 2 consensus sequence for
adenylate kinases is -K-
-
-X-K- where
and X represent hydrophobic and any amino acid, respectively. From
the crystal structure of adenylate kinase, the hydrophobic residues
appear to form a binding pocket for the adenosyl moiety of MgATP, while
NMR measurements indicate that the first lysine in the consensus
sequence could interact with the
- and/or
-phosphate group of
ATP(38) . Lysine 193 in the PRPP synthetases occupies the
cognate position of the second lysine in the adenylate kinase consensus
sequence. The function of the second lysine in the adenylate kinase
segment 2 sequence has not been delineated. It is possible that
Lys
of E. coli PRPP sythetase interacts with
ATP, perhaps by forming hydrogen bonds with either the 2`- or
3`-hydroxyl groups. This is an attractive idea because Lys
reacts with the aldehyde functionalities in oATP that are present
at positions analogous to the 2`,3`-hydroxyl groups of ATP.
Alternatively, Lys
could be interacting with the
triphosphate chain of ATP either directly, or indirectly through the
Mg
cation chelated to the phosphate moieties in the
MgATP complex utilized as a substrate by PRPP synthetase.
In
contrast, Lys is conserved in only four of the PRPP
synthetases. In all other PRPP synthetases, the residue at this
position is an aspartic acid. Interestingly, there are relatively well
conserved lysyl residues present either upstream, downstream, or both
in the other sequences. Of even greater interest, however, is the fact
that the aspartic acid residue (Asp
) found at this
position in human isozyme I is replaced by a histidyl residue in a
mutant PRPP synthetase isolated from patient S. M.(39) . This
mutant enzyme is characterized by greatly decreased sensitivity to
inhibition induced by purine ribonucleoside diphosphates that act both
competitively (ADP) and noncompetitively (ADP, GDP) with respect to
ATP. This raises the intriguing possibility that the residue found at
this position, or alternatively, the sequence around this residue is
somehow involved in controlling inhibition of the enzyme. Kinetic
inhibition studies with PRPP synthetase from S. typhimurium suggested the existence of a second, allosteric site for ADP
binding (8, 9) . Equilibrium dialysis binding studies (7) confirmed that the second site existed and demonstrated
that the allosteric site is available for ADP binding only in the
presence of saturating amounts of the substrates ATP (or the
nonhydrolyzable substrate analog,
,
-methylene ATP) and
Rib-5-P. It was furthermore shown that this site was highly specific
for ADP and did not bind ATP. Our affinity labeling results with the E. coli enzyme imply that Lys
is in the active
site of the enzyme. Lysine 181 could thus be involved in transmitting a
signal to the allosteric site from the active site when it binds
substrates, with the result that the allosteric site becomes competent
to bind inhibitor. Although lysine is replaced by aspartate at the
homologous position in human isozyme I, there is a lysine residue six
amino acid residues upstream of this position that could possibly carry
out a similar function. Alternatively, it is conceivable that
Asp
in the human enzyme functions in a subtly different
way than Lys
does in the E. coli enzyme. It is
interesting in this respect that PRPP synthetases from the enteric
bacteria are insensitive to allosteric inhibition by GDP, whereas the
mammalian and Bacillus subtilis PRPP synthetases are quite
sensitive to GDP inhibition. These GDP-sensitive enzymes all possess
aspartate residues at the cognate position of Lys
in the E. coli enzyme. Thus, differences in the primary structure of
PRPP synthetases in the region around Lys
may play a role
in determining this specificity. Finally, it is possible that this
position represents a junction between the allosteric and the active
sites, controlling inhibitor specificity and sensitivity by virtue of
being shared between the two sites.
It is more difficult to draw
conclusions concerning function about the site of labeling at
Lys and, if it exists, Cys
. These sites are
protected from modification by oATP in the presence of ATP and could
therefore lie in or near the active site. They are found just
downstream of the highly conserved, putative PRPP-binding site (9) in a relatively well conserved region of sequence. Cysteine
at this position is conserved in about 50% of the PRPP synthetases,
while at the Lys
position charged residues and leucine
predominate; lysine is present in only four of the PRPP synthetases.
Segment 3 (Fig. 3) of the adenylate kinase homologous sequences
is also found within the PRPP-binding site sequence. Segment 3 has been
postulated to interact with the triphosphate chain of ATP in these
enzymes(38) . Previous studies with sulfhydryl specific
reagents (40) identified Cys
in S.
typhimurium PRPP synthetase as being very reactive, but possessing
no functional role with respect to substrate binding or catalysis.
Although these considerations make it unlikely that Cys
and Lys
have a specific function in catalysis or
ligand binding in E. coli PRPP synthetase, affinity labeling
results could be explained by these residues lying at the entrance to
the active site and being located in a region that alters conformation
upon substrate binding, such that saturation with ATP prevents reaction
with oATP.
The relationship of the identified lysyl residues in this study to the ``critical lysine'' implicated by pyridoxal phosphate inactivation studies of the S. typhimurium enzyme (24) is not clear. If one assumes that one of our lysines is this critical lysine, ATP protection studies also reported in this study indicating that ATP does not fully protect against inactivation are difficult to reconcile with our studies showing ATP prevents both inactivation and incorporation of oATP. The reason for the discrepancy may lie in the intrinsic differences between the reagents employed. The suggestion that this critical lysine lies in the Rib-5-P subsite may indicate that this residue is not represented among our labeled lysines. Elucidation of these details will require identification of the lysine residue labeled by pyridoxal phosphate.
Although oATP has been assumed to be specific for lysines, our work and the results of Zheng et al.(36) imply that cysteine may be capable of reacting with this reagent as well. The expected reaction product would be a thiohemiacetal. Zheng et al. also identified a cysteinyl residue adjacent to a lysyl residue where both appeared to be labeled, and they postulated that oATP cross-links these residues through reaction of both aldehyde functionalities present in oATP. A similar reaction might occur in our system. Furthermore, although it is often stated that oATP forms a Schiff's base with lysine, there is ample evidence in the literature that this is the exception rather than the rule(26, 35, 36) . Our observations on the lack of effect of reducing agent on the stability of the oATP-enzyme adduct as well as the instability of the adduct to acid pH support this contention.
Finally, ADP is an allosteric inhibitor of PRPP synthetase that binds at a site distinct from the active site, is specific for ADP, and access to which is influenced by substrate saturation of the active site. These circumstances make similar affinity labeling studies probing the allosteric site with oADP an attractive prospect. Studies with this aim are currently underway in our laboratory and may provide insight into the nature of amino acids present in the allosteric binding site. These studies, in combination with x-ray crystallographic studies of B. subtilis PRPP synthetase in progress in our laboratory should produce new insights into the molecular mechanism of catalysis and allostery in PRPP synthetase.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]; Bacillus caldolyticus, X83708[GenBank]; Listeria monocytogenes, M92842[GenBank]; Caenorhabditis elegans, U00036[GenBank]; Synechococcus sp., D14994[GenBank].