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J. Biol. Chem., Vol. 275, Issue 27, 20231-20234, July 7, 2000
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andFrom the Laboratory of Molecular Parasitology and Drug Design, University of North Carolina School of Pharmacy, Chapel Hill, North Carolina 27599-7360
Purine phosphoribosyltransferases
(PRTs)1 of microbes and
mammals are enzymes that catalyze the recovery of preformed bases for
use in cellular metabolism. In free living organisms, purine nucleotides can be generated via de novo synthesis, as well
as by the salvage of preformed bases. In contrast, many parasitic organisms are unable to synthesize purines via de novo
pathways and therefore must rely on the enzymes in salvage pathways,
including PRTs, for the synthesis of purine nucleotides (1). For this reason, enzymes in salvage pathways were proposed more than 30 years
ago as potential targets of therapeutic agents for the treatment of
diseases caused by parasites (2).
Purine PRTs catalyze the reversible transfer of a phosphoribosyl group
from phosphoribosylpyrophosphate (PRPP) to a purine base (adenine,
guanine, hypoxanthine, or xanthine). For those enzymes that have been
studied, the forward reaction appears to be ordered and sequential with
PRPP binding first followed by the purine base (3-5). After catalysis,
pyrophosphate (PPi) is released before the nucleotide.
Reaction chemistry has been reported to proceed via either a
dissociative (Sn1) or an associative
(Sn2) type mechanism (Fig.
1). Based on their similarity to other
enzymes, PRTs have been proposed to catalyze an
Sn1-type reaction with the formation of an
unstable ribooxocarbenium ion intermediate (6, 7), but
Sn2-type chemistry for purine PRTs has not been ruled out (8).
INTRODUCTION
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Fig. 1.
Sn1 versus
Sn2-type reaction mechanisms catalyzed by
hypoxanthine PRTs. The ribooxocarbenium intermediate created in a
two-step Sn1 mechanism is compared with the
predicted transition state for an Sn2-type
mechanism.
Enzymes in salvage pathways that contribute to the synthesis of AMP include adenosine kinase or adenine phosphoribosyltransferase (APRT). Alternatively, adenosine deaminase and a purine nucleoside phosphorylase catalyze the conversion of adenine to hypoxanthine, which may be salvaged by hypoxanthine phosphoribosyltransferase (HPRT). The product, IMP, is a precursor for both AMP and GMP. In most bacteria and nearly all eukaryotes, HPRTs also catalyze the salvage of guanine and, in a few cases, xanthine. In human tissues, substrates available for salvage include hypoxanthine at 8.2 ± 1.3 µM, xanthine at 2.5 ± 0.6 µM, adenine at 0.3 ± 0.15 µM, and adenosine at 0.6 ± 0.2 µM (9). The human HPRT can salvage xanthine, albeit at relatively low levels (10), but instead this base is usually converted to uric acid for excretion as a nitrogenous waste. The identity of the 6-oxopurine substrates salvaged by HPRTs is frequently included in the names of these enzymes, but HPRTs likely are descended from a common ancestral hpt gene of prokaryotes, and substrate specificity can be dramatically altered by single amino acid substitutions (11, 12).
Several bacteria possess two distinct enzymes for the salvage of 6-oxopurines. In those bacteria the primary substrate for the HPRT is hypoxanthine, and guanine is utilized with reduced efficiency (11). The other enzyme, referred to here as xanthine phosphoribosyltransferase (XPRT), has a preference for catalyzing the salvage of guanine and xanthine.
The contributions of specific enzymes to the salvage of purine bases
vary significantly among different organisms. For example, in
Plasmodium falciparum, etiologic agent of the most lethal
form of human malaria, the activity of adenosine kinase is barely
detectable, and APRT activity is 1500-fold below that of HPRT (13). In
this pathogen, adenosine deaminase and purine nucleoside phosphorylase are relatively abundant, suggesting that the major route for the salvage of purine bases leading to both AMP and GMP is through hypoxanthine, which can be converted to IMP by the HPRT-catalyzed reaction.
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Crystal Structures of Purine PRTs |
|---|
For this review, residue numbers refer to the positions of
amino acids in the human HPRT, the bacterial XPRT, or the leishmanial APRT, as these enzymes were the first of their class for which crystal
structures were reported (14-16). Fig. 2
illustrates ribbon diagrams of monomers from these representative
structures, although the enzymes may be functional as dimers or
tetramers. All three of these enzymes possess a core domain composed of
a 4- or 5-stranded parallel 
sheet flanked by 3-4 
helices. The
C-terminal ends of the sheets of the core domains of purine PRTs
form the floor of the active sites of these enzymes. A poorly conserved
hood domain contributes residues that complete the active site and participate in binding purine substrates. For HPRTs and XPRTs the amino
acids that flank the active site are contributed largely by 4 active
site loops (loops I-IV). Residues in the hood domains of HPRTs, XPRTs,
and APRTs come from non-homologous regions of the protein. However, all
three enzymes possess an aromatic residue that forms 
- stacking
interactions with purine substrates (Fig. 3).
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Functional Roles for Conserved Amino Acids |
|---|
Data reported to GenBankTM indicate that the HPRTs of distantly related organisms share extensive primary sequence homology. For example, there is 41% identity for amino acids in the human and a bacterial HPRT (11). However, among well over 20 HPRT sequences reported there are only 9 invariant amino acids and all but the HPRT of Giardia lamblia also are invariant at Glu-133 and Asp-134 (Table I). In general, conserved residues of HPRTs and bacterial XPRTs differ at positions homologous with human Leu-67 and Glu-133 (Ser-36 and Asp-88 in the XPRT of Escherichia coli). Solutions for the crystal structures of HPRTs reveal that the 11 conserved residues immediately flank or are very near the active site of HPRTs (Fig. 3).
|
Among the amino acid sequences deduced from 39 APRT genes reported to GenBankTM, 11 residues are invariant. As shown in Fig. 3, these residues either flank the active site or are located within active site loop II, which is predicted to participate in forming the active site of APRTs during catalysis (16).
If the crystal structures of all purine PRTs are analyzed together with the amino acid sequences reported to GenBankTM, there are only 2 residues (corresponding with human Gly-69 and Asp-134) that are clearly invariant. A G69E mutation virtually inactivates the human HPRT, resulting in Lesch-Nyhan syndrome, whereas a D134G mutation partially inactivates the enzyme, resulting in gouty arthritis (17). The invariant glycine may be essential for the formation of a tight turn and an unusual non-proline cis-peptide in active site loop I of purine PRTs (15, 16, 18-23).
In nearly all purine PRTs, there is a lysine or arginine residue located immediately upstream from the invariant glycine. There have been several suggestions for the functional role of the cis-peptide and the lysine (Lys-68) in active site loop I of HPRTs (20, 23, 24). The presence of the cis-peptide enables the carbonyl oxygen and amide nitrogen, adjacent to the peptide bond, to interact with pyrophosphate atoms and a metal-associated water molecule when they are present in the active sites of HPRTs (21-23, 25). Closed active site structures of HPRTs show that the side chain of Lys-68 forms multiple hydrogen bonds with residues in the opposing subunit in dimers of the enzyme (21, 22, 25). Changes in interactions involving Lys-68, before and after closure of the active site, as the enzyme approaches the transition state of the forward reaction coincide with the weakening or breaking of hydrogen bonds between main chain atoms of loop I residues and ligands in the active site (25). These interactions with active site ligands are replaced by new hydrogen bonds with main chain atoms of residues in active site loop II, which closes over the active site during the transition state. These changes in hydrogen bond interactions with substrates during closure of the active site may facilitate the liberation of pyrophosphate from the active site after catalysis. Directed mutation of Lys-68 to alanine in the human HPRT resulted in the observation of positive cooperativity between subunits in the binding of PRPP, a result that is consistent with loop I being involved in regulating binding interactions with PRPP and pyrophosphate (24). Furthermore, in the HPRT of Tritrichomonas fetus (a protozoan parasite of cattle), the threonine residue at position 68 was proposed to affect the binding of PRPP and PPi to this enzyme (26).
All purine PRTs possess a dipeptide of acidic residues (human HPRT
position 133-134 or leishmanial APRT position 146-147) within a
conserved PRPP binding motif that spans 11 amino acids and includes
residues of active site loop III. Among purine PRTs, the acidic
dipeptide forms hydrogen bonds with the 2'- and 3'-hydroxyls of ribose
moieties of substrates (16, 18, 19, 21, 22, 25, 27). Also, Glu-133 of
HPRTs forms a hydrogen bond with a metal-associated water molecule when
it is present in the active site.
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A Catalytic Base |
|---|
Among HPRTs, the aspartate at position 137 was proposed to play a role in catalysis by interacting with the N-7 position of 6-oxopurine substrates (21, 22, 28). Mutagenesis of the human HPRT indicated that this aspartate likely functions as a catalytic base by facilitating removal of a proton in forward reactions catalyzed by HPRTs (28). Also, hydrogen bonding between this aspartate and the purine N-7 position of a nucleotide analog may contribute to the very tight binding of this inhibitor to HPRTs (21, 22).
The residue at the analogous location in the crystal structure of the
leishmanial APRT is a conserved alanine (Ala-150), suggesting that
either the catalytic mechanism differs between HPRTs and APRTs or that
the catalytic base of APRT is contributed by an amino acid that moves
to participate in the formation of the active site during the
transition state. In HPRTs, active site loop II has been shown to close
over the active site when both substrate ligands are bound and
presumably during the transition state of the reaction (21, 22, 25).
Similarly, the x-ray crystal structure of the leishmanial APRT with
bound AMP hints that residues within active site loop II of this enzyme
may contribute to forming the active site during the transition state
(16). Thus, a catalytic base might be contributed by a residue of
active site loop II of APRTs.
| |
A Site for Binding a Second Metal Ion |
|---|
An aspartate at position 193 of HPRTs has been shown to
participate indirectly in binding pyrophosphate and purines via the formation of a direct protein metal bond with a magnesium ion (21-23,
25) designated M2 (Fig. 4). Asp-193 also
forms hydrogen bonds with two water molecules coordinated by the metal,
and the metal forms coordinated interactions with two oxygens of PRPP or PPi. A third coordinated water molecule forms another
hydrogen bond with the N-3 atom of purine substrates. An invariant
arginine at position 199 of HPRTs participates directly in binding
pyrophosphate (29) and may contribute to positioning both substrates by
being close enough to the carboxyl group of Asp-193 to affect its
position (21-23, 25). Together, these interactions help to position
both substrates for in-line nucleophilic attack at the C1' carbon of PRPP or a nucleotide. A D193N mutation virtually inactivates the human
enzyme resulting in Lesch-Nyhan's syndrome (17). An explanation for
the devastating effects of this mutation is revealed by the existence
of a hydrogen bond between the Asp-193 side chain and the main chain
nitrogen of Asp-196 together with a preference of magnesium for
interacting with oxygen over nitrogen. Thus, the substitution of
asparagine for aspartate at position 193 would disfavor interactions
with both Asp-196 and a magnesium ion.
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|
APRTs possess an invariant aspartate (Asp-44) that flanks the putative PPi binding domain in the active site of the enzyme (Fig. 3). This suggests that a mechanism comparable with that of HPRTs and XPRTs, for binding a second metal and positioning substrates, could possibly exist for APRTs.
As of this writing, the only clear example in eucaryotes of
multiple enzymes for the salvage of 6-oxopurines is in Leishmania donovani (30). In this protozoan parasite there is a gene encoding an "XPRT" as well as an HPRT. The two enzymes appear to be the product of a duplication of the hpt locus with both genes
being expressed in extant parasites. The leishmanial XPRT is novel in its preference for catalyzing the salvage of xanthine over hypoxanthine and guanine. This enzyme differs from all other 6-oxopurine PRTs in
possessing glutamate rather than aspartate at a position homologous with Asp-193 in the human HPRT. This substitution would be predicted to
affect the positioning of the octahedrally coordinated second metal ion
that participates in positioning purine substrates in the active site.
Possibly, the D193E substitution in the L. donovani XPRT is
responsible for the altered substrate specificity of this enzyme.
| |
Roles for Residues in Active Site Loop II of HPRTs |
|---|
As indicated in Table I, Ser-103 and Tyr-104 are invariant in HPRTs and XPRTs. Both are located in loop II, which closes over the active site during the transition state (Fig. 4). A S103R mutation has been shown to impair the human HPRT, resulting in gouty arthritis (17). The main chain carbonyl of Ser-103 forms a hydrogen bond with a pyrophosphate oxygen atom of PRPP, whereas the side chain hydroxyl interacts with a water molecule (Fig. 4, C). This water forms an additional hydrogen bond with a conserved serine (Ser-109), as well as to a 5'-phosphate oxygen of either PRPP or a nucleotide analog in the active site (21, 22, 25). These interactions secure the base of the closed loop II and stabilizes its position over the active site. Like the S103R mutation, a S109L mutation results in partial inactivation of the human HPRT (17). Steady-state kinetic studies of these mutations in the human HPRT (31, 32), together with contemporary structural data, indicate that loop II also contributes to the stable binding of purine substrates. Possibly this is achieved indirectly through van der Waals interactions of loop II residues with conserved aromatic residues (homologous with human Phe-186) that stack above purine substrates.
Analysis of the HPRT from L. donovani revealed that
mutations of the residue homologous with the tyrosine at position 104 of the human HPRT severely reduce the turnover number
(kcat) for the forward reaction catalyzed by the
enzyme (33). The main chain nitrogen of Tyr-104 forms a hydrogen bond
with a pyrophosphate oxygen (Fig. 4), and the side chain hydroxyl forms
a hydrogen bond with a 5'-phosphate oxygen atom of either PRPP or a
nucleotide analog in the active site (21, 22, 25). These interactions position the aromatic ring of Tyr-104 directly above the location of
the glycosidic bond that would be formed or broken during catalysis and
nearly perpendicular to the ribose ring. Thus, the aromatic ring of
tyrosine helps to isolate the reaction center from bulk solvent and
could possibly provide partial electrostatic stabilization for a
positively charged intermediate in an Sn1-type reaction.
| |
Specificity for 6-Oxo- and 6-Aminopurine Substrates |
|---|
An invariant lysine residue at position 165 in the human HPRT
forms a hydrogen bond with the exocyclic oxygen of 6-oxopurine bases
(14). The homologous residue in the HPRT of T. fetus has been demonstrated by site-directed mutagenesis to be the determinant for 6-oxopurine substrate specificity (12). In the only available structure for an APRT, specificity for 6-aminopurines appears to be
determined by the formation of a hydrogen bond with a main chain
carbonyl atom of an arginine residue at position 41 (16).
| |
The Catalytic Mechanism of HPRTs |
|---|
The discovery of two metal ions in the active sites of HPRTs has important implications for the chemistry of the reaction catalyzed by this enzyme. The electron withdrawing potential of the metal ions may contribute to activation of PPi as a leaving group in HPRT-catalyzed reactions. The metal designated M1 forms no direct interactions with active site residues (Fig. 4). However, 4 oxygen atoms belonging to either PRPP or PPi and the ribose moiety of nucleotide substrates (atoms designated O-1, O-2', O-3', O-3B) are within the coordination sphere of this metal (21, 22, 25). In the structure of the trypanosomal HPRT, with PRPP and a purine analog as ligands, the length of the coordinated interaction of M1 with the O-1 atom of PRPP is too long (at 2.6 Å) to satisfy optimally the metal coordination sphere (25). Thus, the electron-withdrawing potential of M1, along with formation of the sixth metal bond to O-1 as the reaction approaches the transition state, may contribute to lowering the activation energy for catalysis.
Previous kinetic studies (6, 7) suggest that HPRTs catalyze an Sn1-type chemical reaction (Fig. 1) where an unstable ribooxocarbenium intermediate would need to be protected from bulk solvent. The deletion of 7 residues from active site loop II of the trypanosomal HPRT does not prevent the enzyme from catalyzing either forward or reverse reactions, albeit at extremely reduced catalytic efficiencies (34). This indicates that total isolation from bulk solvent is not required for the protection of a highly unstable intermediate in the reaction. Furthermore, x-ray crystal structures of the closed active sites of the human, malarial, and trypanosomal HPRTs reveal that the only residue near enough to provide stabilization of a positively charged intermediate is an invariant tyrosine (human Tyr-104), which is located above the ribose ring of bound substrates (21, 22, 25). This suggests that these enzymes could provide only minimal electrostatic stabilization for a ribooxocarbenium intermediate formed during an Sn1-type reaction.
An obvious question that arises from the structure of trypanosomal and
human HPRTs with PRPP bound (24, 25) is why wasn't the PRPP cleaved.
In these structures, PRPP is present in the closed active sites and the
metal (M1) is poised to move closer to the O-1 atom to assist further
in pulling electrons away from the covalent bond that is to be broken.
If Sn1 chemistry were involved, the first half
of the reaction might be expected to occur, resulting in dissolution of
the covalent bond between PPi and the ribose monophosphate.
However, the reaction seems to be awaiting nucleophilic attack by the
purine base, which cannot occur in the reported structures because of
the non-reactive purine analog in the active site. Thus, these crystal
structures provide evidence for the reaction being associative
(Sn2) rather than dissociative
(Sn1).
| |
The Design or Discovery of HPRT Inhibitors |
|---|
Prior to 1999, few HPRT inhibitors had been identified and most yielded Ki values above 10 µM. An exception was a nucleotide analog, carbocyclic GMP, with an IC50 of 0.87 µM versus a mammalian HPRT (35). Recent progress in enzyme structure-based inhibitor design/discovery shows promise that HPRT inhibitors eventually might be developed into drugs for the treatment of diseases caused by parasites. In this regard, the de novo design of mechanistic inhibitors, including transition state analogs and multisubstrate inhibitors, provides an approach to the discovery of potent inhibitors of HPRT activity (36).
Success in the de novo design of a mechanistic type of inhibitor was achieved recently in a series of non-hydrolyzable nucleotide analogs (immucillin 5'-phosphates). As inhibitors of reverse reactions catalyzed by the human HPRT, these compounds yield Ki values in the range of 56-250 nM (37). However, because of "slow onset inhibition," secondary Ki values, determined from the rates of product formation after 2 h in the presence of substrates and inhibitor, were between 1.0 and 14 nM. Although structurally similar to carbocyclic GMP (35), the immucillins differ in that they mimic chemical requirements predicted to exist in the transition state of HPRT-catalyzed reactions (37). Specifically, the immucillins possess nitrogen instead of oxygen in the ring of the ribose moiety and a proton at the N-7 position of the purine moiety. The N-7 proton (predicted to exist for both Sn1- and Sn2-type reaction mechanisms) enables the formation of a hydrogen bond with the invariant Asp-137 residue of HPRTs (21, 22). The nitrogen atom in the ribose ring allows for the formation of a positive charge on the analog of the ribose moiety at physiological pH. In theory, the charge mimics the electrostatic surface potential predicted for a ribooxocarbenium intermediate in Sn1-type reactions. Crystal structures of the human and malarial HPRTs were solved with immucillin inhibitors bound (21, 22). Surprisingly, these structures reveal no strong electrostatic interactions between enzyme residues and the electropositive ribose ring. In explanation, the authors suggest that transition state stabilization is achieved by intramolecular interactions involving the 5'-phosphate of the substrate analog (21). Also, some stabilization could be provided by interactions with the magnesium-coordinated pyrophosphate substrate, whose presence was required to achieve the low Ki values for "slow onset" inhibition (37). Thus, the 2.8-Å hydrogen bond between the N-7 proton of the purine moiety and the invariant aspartate at position 137 (21, 22) as well as altered conformation of the plane of the ribose ring may account for the tight binding characteristics of the inhibitors.
Unfortunately, immucillin 5'-phosphates cannot be tested in their present form as inhibitors of the growth of parasites in vivo because they would have difficulty crossing plasma membranes to get to the target enzyme. Also, an intrinsic potential disadvantage of mechanism-based inhibitors is that that they may target all PRTs that have a similar enzyme mechanism, and it may be more difficult to achieve selectivity for binding to the enzymes of pathogens. Thus, a combined approach of mechanism- and structure-based design may be required to achieve target selectivity in mechanism-based inhibitors.
Computational efforts to identify HPRT inhibitors using the x-ray
crystal structures of target enzymes have been reported. For example,
the rigid body program "DOCK" was used to identify a 240 µM inhibitor of the HPRT from T. fetus (38).
Derivatives of this lead inhibitor were shown to yield
Ki values of 12 µM (38) and 49 nM (39) versus the HPRT from T. fetus. IC50 value determinations with human and
tritrichomonal HPRTs indicate that the compounds selectively inhibited
the enzyme of the parasite. Inhibition of the growth of parasites
in vitro by these compounds was reversed by the addition of
excess purines in the medium, providing evidence that the HPRT was the
most likely target of the inhibitor (38, 39). These are the first
reports in which an HPRT inhibitor has been shown to be effective in
slowing the growth of a protozoan parasite.
| |
Concluding Remarks |
|---|
Purine PRTs have been studied extensively during the latter half
of the twentieth century. A number of crystal structures have been
solved, and considerable data are available from studies of amino acid
substitutions that provide insights into structure/function relationships for this important class of metabolic enzymes.
Mechanistic studies and enzyme structures have been used in the design
and analysis of inhibitors of HPRTs, showing promise that the chemical knock-out of HPRT activity may be achieved within the next few years.
Such inhibitors would represent novel leads for the development of
drugs for the treatment of diseases caused by protozoan parasites.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Richard Wolfenden for advice about Sn1 and Sn2 reactions and Dr. Francisco-Javier Medrano for editorial suggestions, providing alignments for amino acid sequences reported to GenBankTM, and preparing Figs. 2 and 3 for this article.
| |
FOOTNOTES |
|---|
* This minireview will be reprinted in the 2000 Minireview Compendium, which will be available in December, 2000.
To whom correspondence should be addressed: Laboratory of
Molecular Parasitology and Drug Design, Beard Hall, Rm. 326, CB 7360, University of North Carolina School of Pharmacy, Chapel Hill, NC
27599-7360. Tel.: 919-966-6422; Fax: 919-966-0204; E-mail: scraig@unc.edu or eakin{at}unc.edu.
Published, JBC Papers in Press, May 17, 2000, DOI 10.1074/jbc.R000002200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: PRT, purine phosphoribosyltransferase; PRPP, phosphoribosylpyrophosphate; APRT, adenine phosphoribosyltransferase; HPRT, hypoxanthine phosphoribosyltransferase; XPRT, xanthine phosphoribosyltransferase.
| |
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Crystal Structures of Purine...
