(Received for publication, March 7, 1995)
From the
Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase
catalysis and regulation were studied using a new stable carbocyclic
analog of PRPP,
1-
Glutamine phosphoribosylpyrophosphate (PRPP)
The enzymes from Escherichia
coli (Messenger and Zalkin, 1979) and Bacillus subtilis (Wong et al., 1981) have been purified to homogeneity and
characterized (Zalkin, 1993). In addition, 13 glutamine PRPP
amidotransferase sequences have been derived from cloned genes. These
sequences are highly conserved (29-93% identity) leading to the
conclusion that the enzymes in bacteria (Tso et al., 1982;
Makaroff et al., 1983; Gu et al., 1992),
The C-domain contains two nucleotide
sites, an allosteric A site in close proximity to a catalytic C site.
The C site, which binds nucleotide in the crystal structure, is
identified as a catalytic site by virtue of a PRPP-binding sequence
motif (Hershey and Taylor, 1986; Hove-Jensen et al., 1986)
which has also been identified as the active site in two other
phosphoribosyltransferases (Scapin et al., 1994; Eads et
al., 1994). Glutamine PRPP amidotransferase is not only a
glutamine amidotransferase but is also a phosphoribosyltransferase.
PRPP has been modeled into the glutamine PRPP amidotransferase C site
in a position occupied by the ribose-5-phosphate moiety of AMP (Smith et al., 1994). The proximal A site, identified by bound
nucleotide in the crystal structure, is between subunits of the enzyme
tetramer. The proximity of the A and C sites is unusual and may
contribute to the synergistic binding of AMP and GMP that is
responsible for the synergistic end product inhibition (Zhou et
al., 1994).
The mechanism of amide transfer from glutamine is
unknown and it is an open question whether glutamine hydrolysis
precedes, is concerted with, or occurs after attachment of the NH
Figure 1:
A, ribbon diagram of
the glutamine PRPP amidotransferase subunit in the open conformation.
The N-domain with the catalytic residue Cys
Figure 2:
Inhibition by cPRPP. Enzyme activity was
determined as described under ``Experimental Procedures.''
Symbols are
Figure 3:
Calculation of the K for cPRPP.
Enzyme activity was assayed with either 50 µM (
Figure 4:
Alkylation of Cys
Figure 5:
Effect of cPRPP on GMP binding.
Equilibrium dialysis was performed with 1.0 mM cPRPP
(
cPRPP is a stable substrate analog that competes with PRPP
for the glutamine PRPP amidotransferase catalytic site. Interactions
important for binding of PRPP to the C site can be inferred from the
structural model of the homologous B. subtilis amidotransferase (Smith et al., 1994). All amino acids
that interact with PRPP modeled into the C site are conserved in the E. coli enzyme and in all other glutamine PRPP
amidotransferases for which sequences are available. These include
interactions of Lys
The results of experiments reported here support the view that
cPRPP competes with PRPP for the C site. First, enzyme inhibition was
competitive with PRPP in the wild type and in a mutant with a partially
disabled C site having a higher K
By both criteria, cPRPP did not promote formation of
the active conformation. First, binding of cPRPP to the C site did not
activate Cys
There are at least three possibilities to account for the failure of
cPRPP to activate Cys
The best candidate for a hydrogen-bond
donor to the ring oxygen of PRPP is the side chain of Tyr
A third possibility to explain the inability of cPRPP to activate
Cys
The fact that binding of cPRPP to the
C site did not significantly affect nucleotide binding to the A site
has implications for feedback regulation. We favor the explanation that
the small differences in K
The present data for
nucleotide binding in the presence of cPRPP support earlier conclusions
on the binding selectivity of the A and C sites obtained with mutants
(Zhou et al., 1994). With cPRPP bound to the C site, GMP
exhibited a higher affinity than AMP for the A site. Thus, although GMP
and AMP can each bind to both sites, there is a preference of GMP for
the A site and of AMP for the C site. This selectivity combined with
the effect that binding of one nucleotide has on the binding of the
other accounts for the synergistic feedback inhibition of the enzyme by
AMP plus GMP. Although the competition between cPRPP and nucleotide for
the C site did not prevent access of GMP for the A site, binding
affinity is reduced significantly in the absence of synergism.
pyrophosphoryl-2-
,3-
-dihydroxy-4-
-cyclopentanemethanol-5-phosphate
(cPRPP). Although cPRPP competes with PRPP for binding to the catalytic
C site of the Escherichia coli enzyme, two lines of evidence
demonstrate that cPRPP, unlike PRPP, does not promote an active enzyme
conformation. First, cPRPP was not able to ``activate''
Cys
for reaction with glutamine or a glutamine affinity
analog. The ring oxygen of PRPP may thus be necessary for the
conformation change that activates Cys
for catalysis.
Second, binding of cPRPP to the C site blocks binding of AMP and GMP,
nucleotide end product inhibitors, to this site. However, the binding
of nucleotide to the allosteric site was essentially unaffected by
cPRPP in the C site. Since it is expected that nucleotide inhibitors
would bind with low affinity to the active enzyme conformation, the
nucleotide binding data support the conclusion that cPRPP does not
activate the enzyme.
(
)amidotransferase catalyzes the initial reaction in de novo purine nucleotide synthesis and is the key regulatory
enzyme in the multistep pathway to AMP and GMP. Similar to other
glutamine amidotransferases (Zalkin, 1993), NH
can replace
glutamine as amino donor in vitro (Messenger and Zalkin, 1979)
and in vivo (Mäntsälä and Zalkin, 1984a; Mei
and Zalkin, 1990). The reactions are glutamine + PRPP
PRA
+ glutamate + PP
and NH
+ PRPP
PRA + PP
.
(
)lower eukaryotes
(
)(Mäntsälä and Zalkin, 1984b; Ludin et al., 1994), invertebrates (Clark, 1994), vertebrates
(Brayton et al., 1994; Zhou et al., 1990; Iwahana et al., 1993) and plants (Ito et al., 1994; Kim et al., 1995) are homologous and are structurally similar. The
glutamine PRPP amidotransferases from E. coli and B.
subtilis are thus viewed as models for the enzyme from other
organisms. The x-ray structure of the B. subtilis glutamine
PRPP amidotransferase with bound nucleotide has been solved recently
(Smith et al., 1994). The enzyme contains an
NH
-terminal glutamine-binding domain (N-domain) that is
joined to a COOH-terminal domain (C-domain) with sites for
NH
-dependent synthesis of PRA and for end product
inhibition by AMP and GMP. The N-domain has sequence and presumably
structural similarity to the glutamine-binding N-domains of asparagine
synthetase and glucosamine 6-phosphate synthase, enzymes that belong to
a ``purF'' subfamily of amidotransferases (Zalkin, 1993). For
this subfamily the N-domain contains an NH
-terminal active
site cysteine residue that is essential for transfer of the glutamine
amide to the second substrate.
group to C-1 of PRPP. In the crystal structure of the inhibited B. subtilis glutamine PRPP amidotransferase containing bound
AMP, the catalytic thiol of Cys
in the N-domain and C-1 of
PRPP modeled into the C-domain are separated by a 16-Å
solvent-filled space (Fig. 1). There are thus at least two
barriers to catalysis. First, the inhibited enzyme is in a conformation
described as ``open'' in which the glutamine and PRPP sites
are too far apart for catalysis. Second, although Cys
is
proximal to the interdomain boundary, its sulfhydryl group is
sequestered in a cavity within the N-domain where it is inaccessible to
glutamine. Given the very low rates of glutamine hydrolysis and
reaction with glutamine affinity analogs in the absence of PRPP
(Messenger and Zalkin, 1979), the latter substrate must somehow
``activate'' Cys
for the initial steps of
glutamine amide transfer.
is shown at the
top of the diagram and the C-domain with PRPP modeled into the C-site
at the bottom. The catalytic thiol of Cys
and C-1 of PRPP
are 16-Å apart.
strands are depicted as arrows,
-helices as spirals, and other structures as a tube. Side chain atoms are shown for Cys
,
Arg
, and Tyr
. A 30-residue loop, which would
obscure the PRPP site but does not contact it, is omitted for clarity. B, close-up view of the active site. The view is the same as
in A with additional side chains from the PRPP-binding
sequence fingerprint (Asp
, Asp
,
Ser
, Ile
, Val
,
Arg
, and Gly
) included. Atom coloring in
both A and B: C, black; O, red; N, blue; S, yellow;
and P, green. Diagrams were made with the program
Molscript (Kraulis, 1991).
Due to chemical lability it has not been
possible to investigate how binding of PRPP to the glutamine PRPP
amidotransferase C site activates Cys for catalysis and
what effect this interaction has on binding of nucleotides to the A and
C sites. The present approach was to utilize a new stable PRPP analog,
cPRPP, to further examine the geometry of the C site, the requirements
for activation of Cys
for amide transfer and the effect
that a substrate analog has on nucleotide binding. The results
demonstrate that cPRPP competes with PRPP for the C site and in
addition can block binding of nucleotide to the C site. Yet, unlike
PRPP, cPRPP does not activate Cys
for catalysis.
Furthermore, a mutation that reduces the affinity of nucleotide for the
C site has no appreciable affect on PRPP or cPRPP binding, thus
supporting the assignment of distinct ribose phosphate and purine base
binding regions in the C site (Smith et al., 1994).
Synthesis of cPRPP
The PRPP analog cPRPP was
synthesized as reported (Parry and Haridas, 1993). In cPRPP a methylene
carbon replaces the ring oxygen of PRPP. The compound was racemic
because the starting materials employed in the synthesis were not
asymmetric, and no resolutions were carried out. The compound used in
these studies therefore consists of a mixture of equal parts of the two
possible enantiomers, one of which corresponds in absolute
configuration to the configuration of natural PRPP.
Enzyme Purification
Wild type E. coli glutamine PRPP amidotransferase and the P410W C site mutant enzyme
were overproduced from plasmid pGZ14 and its mutant derivative,
respectively, in E. coli strain TX358 (purF recA) as
described (Zhou et al., 1993). Cells were harvested in late
log phase from cultures grown in 2-liter flasks and were stored at
-20 °C prior to purification. The enzyme was purified (Mei
and Zalkin, 1989) to approximately 95% homogeneity as judged by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli, 1970).
Enzyme at a concentration of approximately 20 mg/ml was stored at
-20 °C. Protein concentration was determined by A using the value 8.12 for a 1% solution (Messenger and Zalkin,
1979). Specific activity was typically 15-20 units/mg protein for
the wild type enzyme and 7.5 units/mg protein for the P410W enzyme. A
unit of activity is defined as the amount of enzyme needed to produce
1.0 µmol of glutamate/min.
Enzyme Assay
Glutamine-dependent activity was
determined by measurement of the glutamate produced (Messenger and
Zalkin, 1979). Reactions contained 0-0.5 mM PRPP
(Sigma), 10 mM glutamine, 5 mM MgCl,
0-1.0 mM cPRPP, 50 mM Tris-HCl (pH 8.0) and
approximately 40 ng of enzyme in a total volume of 0.1 ml. Incubation
was at 37 °C, usually for 10 min. Reactions were quenched in a
boiling water bath for 30 s, and glutamate was determined by the
glutamate dehydrogenase method (Messenger and Zalkin, 1979).
Glutaminase activity in the absence of PRPP was determined by a similar
procedure, but the enzyme concentration was increased 10-100-fold
due to the low rate of reaction and samples were removed at timed
intervals to accurately determine the rate of glutamate production. All
assays were shown to be linear for at least 12 min, and rates were
proportional to enzyme concentration over at least a 4-fold range.
Inactivation by DON
Enzyme (approximately 8
µg) was incubated at room temperature for 0-30 min in a
mixture containing 1 mg/ml bovine serum albumin, 50 mM potassium phosphate (pH 7.5), 10 µM DON (Sigma) if
present, 1.0 mM PRPP if present, and 1.0 mM cPRPP, if
present, in a total volume of 0.1 ml. At specific times, 5-µl
aliquots were removed and enzyme activity remaining was determined in
the standard 0.5-ml assay for glutamate production (Messenger and
Zalkin, 1979). The 100-fold dilution quenched the reaction of enzyme
with DON.
Nucleotide Binding
Nucleotide binding was
determined by equilibrium dialysis (Zhou et al., 1994) using
chambers of 100 µl. Chambers were separated by a
12,000-14,000 molecular weight cut off dialysis membrane
(Spectrapore) that was stored in 200 mM Tris-HCl (pH 7.5), 20
mM MgCl, and 0.01% sodium azide at 4 °C. The
membrane was blotted on filter paper before loading between the
chambers. One chamber contained 200 mM Tris-HCl (pH 7.5), 20
mM MgCl
, 28 nM
[2,8-
H] AMP, or 35 nM
[8-
H]GMP (0.1 µCi), 0-4.6 mM
unlabeled AMP or GMP, and 1.3 or 2.0 mM cPRPP, if present, in
a total volume of 50 µl. The other chamber contained 10 mM Tris-HCl (pH 7.5) and 200-300 µM enzyme subunit
in a total volume of 50 µl. Radioactive purine nucleotides were
purchased from Moravek Biochemicals, Inc. Dialysis was for 20 h at 4
°C in a rotating apparatus. Samples of 40 µl were removed from
each chamber and were counted for radioactivity.
Data Analysis
Data for substrate saturation by
PRPP were fit to the Michaelis-Menten equation using Ultrafit software
(Biosoft, Cambridge, United Kingdom). K was also calculated by the graphical method of Dixon (Dixon
and Webb, 1979) using Ultrafit. Equilibrium binding data were fitted to
the Scatchard equation (Scatchard, 1949) by non-linear regression using
Ultrafit.
Inhibition by cPRPP
cPRPP was evaluated as a
PRPP analog for glutamine PRPP amidotransferase. The data in Fig. 2show that inhibition of the wild type enzyme by cPRPP was
competitive with PRPP. The K for PRPP of
53 ± 12 µM agrees with the value of 67 µM determined previously (Messenger and Zalkin, 1979). Inhibition
data analyzed graphically by the method of Dixon (Dixon and Webb,
1979), shown in Fig. 3, yield a K
of 116 ± 25 µM for cPRPP. Since the
cPRPP preparation is a 1:1 racemic mixture (Parry and Haridas, 1993),
the actual K
for cPRPP is 58 ± 13
µM, a value that is similar to the K
for PRPP. We also examined the inhibition of a C site mutant
enzyme by cPRPP. The P410W replacement in the C site decreases the
binding affinity of AMP for the C site by 5-fold with a corresponding
decrease in enzyme inhibition by AMP (Zhou et al., 1994).
Inhibition of P410W by cPRPP was competitive with PRPP (data not
shown). The K
of PRPP was 31 ± 5
µM and the K
for cPRPP was
53 ± 5 µM, values not significantly different than
those obtained for the wild type. Thus, cPRPP competes effectively with
PRPP for the amidotransferase C site.
, no added cPRPP;
, 0.1 mM cPRPP
added. Data were plotted using the Lineweaver-Burke equation. V is in units/mg protein.
) or
100 µM (
) PRPP and varied cPRPP. Data were plotted
using the graphical method of Dixon(17) . V is in
units/mg protein.
Glutamine Hydrolysis
Synthesis of
phosphoribosylamine is tightly coupled to glutamine hydrolysis. In
contrast, some amidotransferases catalyze a glutaminase activity in
which glutamine hydrolysis can be uncoupled from product formation
(Zalkin, 1993). For example, glutamine hydrolysis in anthranilate
synthase, a ``trpG'' subfamily amidotransferase, is dependent
upon chorismate, the second substrate, and is assayed in the presence
of EDTA which blocks synthesis of anthranilate (Nagano et al.,
1970). In cases where the rate of glutaminase activity is comparable to
the overall rate of product formation, such as in the reaction
catalyzed by anthranilate synthase, glutamine hydrolysis may reflect a
step in the overall reaction. An active site cysteine is required for
reaction of glutamine. For glutamine PRPP amidotransferase, the rate of
PRPP-independent glutaminase activity was reported to be about 4% of
the overall rate (Messenger and Zalkin, 1979). The limited capacity for
glutamine hydrolysis in the absence of PRPP can now be explained by the
finding of an inactive conformation for the nucleotide-inhibited enzyme
(no PRPP in the C site) in which Cys is sequestered and is
inaccessible to glutamine (Smith et al., 1994). This
information strongly implies that binding of PRPP to the C site is
needed to ``activate'' Cys
. The effect of PRPP on
glutaminase activity could not be determined previously since there was
no way to uncouple glutamine hydrolysis from phosphoribosylamine
production. We have therefore reinvestigated the glutaminase activity
to determine whether cPRPP bound to the PRPP site can activate
Cys
for glutamine hydrolysis. Data summarized in Table 1show a basal rate of PRPP-independent glutaminase that was
0.8% of the overall rate for reaction of glutamine with PRPP. This low
rate of glutaminase was not stimulated by cPRPP. Ribose 5-P, on the
other hand, stimulated the rate of glutaminase by 10-fold, as had been
observed earlier (Messenger and Zalkin, 1979).
Affinity Labeling of Cys
Cys is an active site residue required for glutamine amide transfer.
Affinity labeling of Cys
with the glutamine analog DON is
presumed to mimic the nucleophilic attack of Cys
on the
carboxamide of glutamine, resulting in release of ammonia and formation
of a hypothetical
-glutamyl-enzyme covalent intermediate (Nagano et al., 1970; Zalkin, 1993). Nucleophilic attack of Cys
on the
-carbon of DON results in displacement of molecular
dinitrogen and alkylation of Cys
. The data in Fig. 4show, as reported previously (Messenger and Zalkin, 1979),
that affinity labeling of Cys
by DON was dependent upon
PRPP. cPRPP did not support the reaction of DON with Cys
,
confirming that cPRPP did not activate Cys
.
by DON.
Enzyme was incubated with DON, as described under ``Experimental
Procedures.'' Reactions with PRPP are shown by (
), those with
no PRPP, with cPRPP, or a control that did not contain DON, are all
shown by (
). Samples were removed from each incubation and were
assayed for activity.
Effect of cPRPP on Nucleotide Binding
Glutamine
PRPP amidotransferase is subject to allosteric end product inhibition
by adenine and guanine nucleotides. AMP and GMP can each bind to two
sites/subunit, the allosteric A site and catalytic C site (Zhou et
al., 1994). Synergistic inhibition by AMP plus GMP results from
synergistic binding of AMP to the C site and GMP to the A site.
Availability of the stable competitive inhibitor cPRPP affords the
opportunity to examine directly the expected competition between
substrate analog and nucleotide for the C site and the consequences
that cPRPP binding to the C site has on nucleotide binding to the A
site. The lability of PRPP has precluded its use in equilibrium binding
experiments. Equilibrium binding of GMP to the enzyme is shown in Fig. 5. GMP binding extrapolated to 1.7 eq nucleotide/subunit
with a K of 220 µM (Table 2). In contrast to earlier experiments (Zhou et
al., 1994), the K
values of GMP for
the A and C sites were not sufficiently different to be resolved. We
infer that GMP was bound equally to the A and C sites (Table 2).
In the presence of cPRPP the binding stoichiometry was reduced to 0.90
eq GMP/subunit. It is therefore reasonable to infer that binding of
cPRPP to the C site blocked nucleotide binding to this site and
marginally decreased the affinity of GMP for the A site. In a similar
manner, cPRPP blocked binding of AMP to the C site (Table 2). In
this case, however, perhaps as a result of an increased K
, binding of AMP to the A site was not
detected. Under the experimental conditions employed, binding of a
ligand with a K
> 1.0 mM would not be detected.
) or without cPRPP (
). Enzyme concentration was 84.25
µM (337 µM subunit).
To reduce the potential ambiguity of
assigning bound nucleotide to A and C sites, we examined the effect of
cPRPP on nucleotide binding to the P410W mutant. This C site
replacement effectively decreases, below the limit of detection, the
capacity of a single nucleotide to bind to the C site (Zhou et
al., 1994). However, approximately 1 eq of GMP could bind to the A
site of this mutant and thus permitted synergistic binding of AMP to
the mutant C site with a 5-fold increased K compared to the wild type. This suggests that the binding
affinity for nucleotide to the mutant C site is reduced about 5-fold.
Data in Table 2, line 5, show binding of 0.83 eq of GMP to the
P410W mutant enzyme. The bound GMP is assigned to the A site by virtue
of the preference of GMP for the A site (Zhou et al., 1994)
and the disabling mutation in the C site. Binding of GMP to the P410W A
site was unperturbed by cPRPP (Table 2, line 6). Given the
competitive inhibition and unchanged K
for cPRPP, it is safe to infer saturation of the P410W C
site by cPRPP. We next determined the capacity of GMP to bind to the A
site under conditions that mimic saturation of the enzyme with the two
substrates. For this purpose Cys
was alkylated with the
glutamine affinity analog DON until less than 3.2% of the
glutamine-dependent activity remained. The enzyme was dialyzed to
remove PRPP and residual unbound DON, and GMP binding was determined in
the presence or absence of cPRPP. Data in Table 2, lines 7 and 8,
show that the two substrate analogs had no significant effect on
binding of GMP to the A site. The implications of this result on the
capacity of these substrate analogs to promote an active enzyme
conformation and influence nucleotide binding are discussed below.
with the pyrophosphate
-phosphate, Arg
with the 5-phosphate, and the 2- and
3-hydroxyls of PRPP with a Mg
ion. In addition,
Thr
, Asp
, and Asp
, all
numbered according to the E. coli sequence, and a water
molecule contribute to the octahedral ligand field of the
Mg
. PRPP has been modeled into the C site with a
C3-endo pucker to conform with the conformation of the ribose phosphate
moiety of bound AMP (Smith et al., 1994). Although it is not
known whether the conformations of cPRPP and PRPP are identical, it is
reasonable to expect the same groups to be involved in binding the
substrate and substrate analog to the glutamine PRPP amidotransferase C
site.
for
nucleotides. The K
for cPRPP was not
significantly different in the wild type and mutant enzymes, and these
values were similar to the K
for PRPP in
each case. Second, binding of cPRPP to the C site excluded binding of
nucleotides to this site. Given these results we determined whether
binding of cPRPP to the C site could promote formation of an active
``closed'' conformation of the enzyme. Two features are
expected to distinguish closed and open conformations. (i) The ability
of Cys
to participate in glutamine hydrolysis and amide
transfer will differ. In the feedback-inhibited inactive open
conformation seen in the crystal structure, Cys
is
sequestered (Smith et al., 1994) and is inaccessible to
glutamine or DON, a glutamine affinity analog. Additionally, in this
open conformation, the positions of Cys
and bound PRPP are
too far apart for N-transfer (Smith et al., 1994). Our working
hypothesis is that PRPP is required both to activate Cys
for reaction with glutamine or DON by making it sterically
accessible and to more favorably position the glutamine and PRPP sites
for amide transfer. (ii) Nucleotide binding to closed and open enzyme
conformations should differ. Based on the inaccessibility of Cys
and its separation from the PRPP-binding site, it appears
unlikely that substrates can bind with high affinity to the inhibited
enzyme having nucleotides in the A and C sites (Smith et al.,
1994). Nucleotide bound to the allosteric A site which is between
subunits of the tetrameric enzyme is likely responsible for the open
conformation of the active site seen in the crystal structure of the
inhibited enzyme. Likewise, nucleotides are expected to have a low
binding affinity for the enzyme-substrate complex in the closed active
conformation.
for reaction with glutamine or DON. Second,
although binding of cPRPP to the C site excluded binding of AMP to this
site in the wild type enzyme, binding of AMP or GMP to the A site was
not perturbed in the wild type or in the P410W C site mutant,
suggesting that the cPRPP
enzyme complex was not in the active
conformation. These results have implications for understanding
catalysis and inhibition by nucleotides and are discussed below.
for reaction with glutamine and DON.
First, cPRPP may assume a conformation different from that of PRPP, and
consequently binding to the C site may not be identical for the
substrate and substrate analog. This could result in different
ligand-protein interactions that might account for the different
capacities to activate. The competitive kinetics for cPRPP inhibition
and the similarity of K
for PRPP and K
for cPRPP argue against, but do not
eliminate, this possibility. Second, a structural difference between
PRPP and cPRPP could prevent the conformational transition between open
and closed states of the enzyme. cPRPP does not contain the furanose
ring oxygen atom that is present in PRPP. A specific interaction of the
furanose ring oxygen of PRPP with the protein may contribute to the
conformational change needed to activate Cys
. This would
explain the limited capacity of ribose 5-phosphate but not cPRPP to
activate Cys
. Whether altered ligand-protein interactions
result from different cPRPP conformations or different functional
groups cannot be distinguished presently and must await
crystallographic analysis.
,
a conserved residue in all glutamine PRPP amidotransferase sequences.
In the crystal structure Tyr
is positioned close to the
furanose ring oxygen of the ribose moiety of AMP in the C site or of
PRPP modeled into this site (Fig. 1). Tyr
is
connected directly to the cavity in which Cys
is
sequestered via Arg
, a residue that lines one wall of the
cavity and is also invariant. A hydrogen bond between the Tyr
hydroxyl and the ring oxygen of PRPP may facilitate a structural
change that unmasks Cys
for reaction with glutamine.
Affinity labeling and mutagenesis experiments support a role for
Tyr
in glutamine amide transfer. Tyr
was one
of the residues affinity labeled by
5`-p-fluorosulfonylbenzoyladenosine (Zhou et al., 1993). Labeling by 5`-p-fluorosulfonylbenzoyladenosine
bound to a nucleotide site places Tyr
in proximity to the
C site. Replacement of Tyr
by Ala resulted in an enzyme
that retained 75% of the wild type activity using NH
as
substrate but retained only 1% activity with glutamine. Thus Tyr
is needed not for binding of PRPP to the C site, but rather for
glutamine amide transfer. An interaction of Tyr
with the
ring oxygen of PRPP may contribute to activation of Cys
.
is that the reactivity at C1 of PRPP is important for
the activation of Cys
and amide transfer from glutamine. If
formation of a hypothetical Cys
-glutamine tetrahedral
intermediate were in some way coupled to the availability of a site to
accept the amide, cPRPP would not serve this function and reaction of
Cys
with glutamine or DON would not occur. cPRPP, unlike
PRPP, is not subject to nucleophilic attack at carbon 1 with
displacement of pyrophosphate.
for nucleotide
binding to the A site in the presence and absence of cPRPP indicate
that binding of cPRPP did not result in the closed enzyme conformation
having low affinity for nucleotide binding. We consider unlikely the
possibility that nucleotides bind with similar affinity to the A site
in active closed and inactive open conformations. In this light there
are three conclusions to be derived from the data in Table 2.
First, alkylation of Cys
with DON which mimics the inferred
enzyme-glutamate covalent intermediate in catalysis (Zalkin, 1993) did
not assist formation of the closed conformation having low affinity
nucleotide binding to the A site, in the presence or absence of cPRPP.
Thus, binding of PRPP, not glutamine, appears to be the key step for
enzyme activation. Second, the K
for
binding of GMP to the A site was similar in the wild type, with or
without cPRPP in the C site, and in the P410W enzyme with or without
cPRPP in the C site. Thus, GMP binding to the A site is not dependent
on nucleotide in the C site. Finally, the P410W mutation is seen to
reduce the affinity of nucleotide but not cPRPP for the C site. This is
consistent with the x-ray structural model of the C site in which PRPP
binds to the position occupied by the ribose phosphate moiety of the
nucleotide (Smith et al., 1994).
-amine;
cPRPP, 1-
-pyrophosphoryl-2-
,
3-
-dihydroxy-4-
-cyclopentanemethanol-5-phosphate; DON,
6-diazo-5-oxo-L-norleucine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.