Converting the Guanine Phosphoribosyltransferase from Giardia lamblia to a Hypoxanthine-guanine Phosphoribosyltransferase*

Guanine phosphoribosyltransferase fromGiardia lamblia, a key enzyme in the purine salvage pathway, is a potential target for anti-giardiasis chemotherapy. Recent structural determination of GPRTase (Shi, W., Munagala, N. R., Wang, C. C., Li, C. M., Tyler, P. C., Furneaux, R. H., Grubmeyer, C., Schramm, V. L., and Almo, S. C. (2000)Biochemistry 39, 6781–6790) showed distinctive features, which could be responsible for its singular guanine specificity. Through characterizing specifically designed site-specific mutants of GPRTase, we identified essential moieties in the active site for substrate binding. Mutating the unusual Tyr-127 of GPRTase to the highly conserved Ile results in 6-fold lower K m for guanine. A L186F mutation in GPRTase increased the affinity toward guanine by 3.3-fold, whereas the corresponding human HGPRTase mutant L192F showed a 33-fold increase in K m for guanine. A double mutant (Y127I/K152R) of GPRTase retained the improved binding of guanine and also enabled the enzyme to utilize hypoxanthine as a substrate with a K m of 54 ± 15.5 μm. A triple mutant (Y127I/K152R/L186F) resulted in further increased binding affinity with both guanine and hypoxanthine with the latter showing a lowered K m of 29.8 ± 4.1 μm. Dissociation constants measured by fluorescence quenching showed 6-fold tighter binding of GMP with the triple mutant compared with wild type. Thus, by increasing the binding affinity of 6-oxopurine, we were able to convert the GPRTase to a HGPRTase.

Giardia lamblia is an anaerobic binucleate flagellated protozoan causing intestinal infection in mammals (14). Two separate parallel pathways providing the major means of synthesizing AMP and GMP are catalyzed by adenine phosphoribosyltransferase and guanine phosphoribosyltransferase (GPRTase), respectively, constituting the primary routes of purine salvage in this organism (15). The lack of interconversion between AMP and GMP has rendered either of the two purine salvage enzymes potential targets for antigiardiasis chemotherapy (1,15).
G. lamblia GPRTase shows little sequence homology with human hypoxanthine-guanine phosphoribosyltransferase (HG-PRTase) and other known purine PRTases (16). It converts only guanine to its corresponding nucleotide with an unusually high K m of 16.4 Ϯ 1.8 M and a very high k cat of 76.7 Ϯ 2.5 s Ϫ1 compared with those of other known purine PRTase-catalyzed reactions (5). It exhibits a very low catalytic activity with hypoxanthine as substrate (K m Ͼ200 M) and has no apparent binding with xanthine at all. It is the only specific GPRTase, as far as we are aware, that has been thoroughly identified thus far.
The crystal structure of G. lamblia GPRTase has recently been solved at a 1.75-Å resolution (12). The enzyme protein, complexed with a transition state GMP analog, immucillin G phosphate together with Mg 2ϩ and pyrophosphate, showed the presence of a common Rossman's fold and the typical hood domain present among all type I PRTases (7)(8)(9)(10)(11)(12)(13). Although primary interactions between the purine substrate and the amino acid residues at the active site are highly conserved among all the purine PRTases with known crystal structures (7,11,17), minor structural differences in the hood region have been noted between the GPRTase and human HGPRTase (7). In Giardia GPRTase, the backbone carbonyls of Asp-181 and Asp-187 interact with the exocyclic N2 of guanine, with estimated distances of 2.7 and 3.1 Å, respectively (12). The purine ring is stacked between two aromatic residues, Trp-180 and Tyr-127. Again, the hydrophobic ring stacked on top of the purine ring has been well conserved among all the PRTases. A similar Trp residue is present in Toxoplasma gondii HGX-PRTase (10), whereas a Tyr residue is present at the corresponding position in Tritrichomonas foetus HGXPRTase (13) and a Phe residue in human HGPRTase (7). The presence of Tyr-127 in the GPRTase is, however, highly unusual, because a well conserved Ile or Leu residue has been identified at the corresponding position among all the other PRTases (7). It appears from the three-dimensional structure of GPRTase (see Fig. 1A) that Tyr-127 could cause extra steric hindrance to purine binding compared with the corresponding Ile-135 in human HGPRTase (Fig. 1B).
Another well conserved residue in the purine-binding pocket * This work was supported in part by National Institutes of Health Grant AI-19391. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 1 The abbreviations used are: PRTase, phosphoribosyltransferase; GPRTase, guanine phosphoribosyltransferase; HG(X)PRTase, hypoxanthine-guanine-(xanthine) phosphoribosyltransferase; PRPP, ␣-D-5phosphoribosyl-1-pyrophosphate; PP i , inorganic pyrophosphate. among type I purine PRTases is the Leu-192 in human HG-PRTase showing a close nonpolar association with the C2 end of bound purine (7). This particular interaction is apparently so specific that a simple L192I mutation in the human enzyme resulted in a 16-fold higher apparent K m for guanine (23). It is not known if a similarly specific interaction exists between the bound purine and the corresponding residue Leu-186 in Giardia GPRTase, or if the relatively high K m for guanine in the GPRTase-catalyzed reaction could be attributed to a lack of such specific association.
The conserved Lys residue, which interacts with the exocyclic O6 of bound purine through hydrogen bonding in all known structures of purine PRTases, is also present in Giardia GPRTase as Lys-152 (Fig. 1A). However, although the ⑀-NH 2 group of Lys residue in human HGPRTase (7), T. foetus HGX-PRTase (13) and other H/G/XPRTases (8,9,11) is invariably within 3 Å from O6 of the bound purine, the corresponding Lys-152 in GPRTase is 6.3 Å away from the O6 group of bound guanine moiety with two ordered water molecules in between. We reasoned that this apparently much weaker bonding between the O6 of purine and Lys-152 may have provided the basis why 6-oxopurines other than guanine cannot be substrates for the GPRTase-catalyzed reaction. Guanine binding to GPRTase is apparently dependent primarily on the interactions of backbone carbonyls of Asp-181 and Asp-187 with the exocyclic N2 of guanine (12). These intriguing structural features have led us to designing, generating, and characterizing site-specific mutants of GPRTase and comparing them with the corresponding mutants of human HGPRTase for elucidating the roles of individual residues in the purine binding region that determine purine specificity. In a final triple mutant (Y127I/K152R/L186F) of the GPRTase, both guanine and hypoxanthine can be used as substrates, indicating a successful general increase in 6-oxopurine binding affinity in the enzymecatalyzed reaction.

MATERIALS AND METHODS
Chemicals and Reagents-All the chemicals used in present studies, including hypoxanthine, guanine, xanthine, adenine, IMP, GMP, XMP, AMP, PP i , and the tetrasodium salt of PRPP, were purchased from Sigma and are of the highest purity available. Xanthine oxidase (1 unit/mg of protein) was from Roche Molecular Biochemicals, and guanase (0.1 unit/mg of protein) was from Sigma.
Enzyme Purification-Recombinant G. lamblia GPRTase, cloned in a pBAce plasmid and expressed in transfected Escherichia coli S606 (⌬gpt-pro-lac, thi, hpt, Rec A Ϫ ), was purified to homogeneity as described previously (16). The purified enzyme samples were stored at Ϫ80°C with no detectable loss of activity after 4 months. The recombinant human HGPRTase was purified from E. coli S606, transformed with pBAcprt expression vector, by a previously described procedure (18).
Enzyme Assays-Kinetic data of enzyme-catalyzed reactions were collected using a Beckman DU-640 spectrophotometer equipped with a Peltier temperature controller. The formation of IMP and GMP were followed spectrophotometrically at 245 and 257.5 nm, respectively. All measurements were carried out in 100 mM Tris-HCl, pH 7.5, and 12 mM MgCl 2 at 37°C (5). Under these conditions, the changes in extinction coefficients for formation of IMP from hypoxanthine and GMP from guanine were 1770 and 6000 M Ϫ1 cm Ϫ1 , respectively (19). The final volume of assay mixture, containing various amounts of substrates, was 0.5 ml. All data points were completed in triplicate, and the average values were reported.
Data Analysis-Initial rate data were fitted into Equation 1 for a sequential mechanism described by Cleland (19) using kinetic software from Bio-Metallics, Inc. (K⅐CAT) and IntelliKinetics (KinetAssyst II). Kinetic constants were calculated using a weighted linear regression. Nomenclatures are that of Cleland (19): v, initial velocity; V max , maximum velocity; A, concentration of substrate A; B, concentration of substrate B; S, substrate concentration; K m , apparent Michaelis con- Equilibrium Dissociation Constant-The dissociation constants of GMP for binding to wild type and mutant GPRTases were determined by following the quenching of intrinsic fluorescence of Trp-180 in the enzyme protein. Fluorescence was measured using a LS 50B fluorometer (PerkinElmer Life Sciences). The excitation wavelength used was 297 nm, and the emission was monitored from 300 to 400 nm. Fluorescence intensity was measured at different ligand concentrations, and the fractional saturation (F) was calculated at each ligand concentration. The dissociation constant was obtained using Equation 2.
F m is the factor allowing for maximum saturation.
Site-directed Mutagenesis-Site-directed mutagenesis of G. lamblia GPRTase and human HGPRTase genes were performed using the Quikchange kit from Stratagene. Oligonucleotide primers were designed and synthesized to generate specific mutants. Following the polymerase chain reaction, mutant plasmid was transformed into E. coli S606. Plasmid DNA isolated from the transformants were sequenced for verification, and the recombinant mutant protein was then purified from the transformed E. coli lysate using the same procedure as for the wild-type enzyme purification (16,18). The stability of each mutant enzyme activity was tested in repeated assays. There was no detectable difference found between the wild type and the mutants.
Complementation Analysis-Wild-type and specific site-directed mutant expression plasmids were transformed into E. coli strain S609 that has the de novo synthesis enzymes and HPRTase and GPRTaseencoding genes mutated or deleted (ara, ⌬pro-gpt-lac, thi, hpt, pup, purH, J, strA) (20). Cell colonies, grown on LB plates with 100 g/ml ampicillin and 50 g/ml streptomycin, were picked and inoculated into LB medium containing both of the antibiotics. A 1% inoculum of the overnight culture was used for growing various S609 transformants in the synthetic low phosphate induction medium (21) in the presence of either 0.2 mM guanine or 0.2 mM hypoxanthine. Growth after 16 h was monitored by light absorbance at 600 nm.

RESULTS AND DISCUSSION
The crystal structure of the active site in G. lamblia GPRTase bound to immucillin G phosphate, Mg 2ϩ , and PPi ( Fig. 1A) shows several apparent distinctions from, as well as some similarities with, the human HGPRTase active site complexed with GMP and Mg 2ϩ (Fig. 1B). Tyr-127, stacked under guanine in GPRTase, appears to be oriented at a perpendicular angle with respect to the bound purine ring and may not accommodate guanine binding as well as the corresponding Ile-135 in the human enzyme. Leu-186 in GPRTase interacts with the C2 end of the bound guanine moiety similarly to Leu-192 in human HGPRTase. The distance between Lys-152 and the exocyclic O6 of bound guanine in GPRTase is much too far compared with the distance between Lys-165 and guanine O6 in human HGPRTase. These residues in GPRTase and the Leu-192 residue in the human enzyme were subjected to sitedirected mutagenesis for structure-function analysis and comparison. Asp-187 in GPRTase and Asp-193 in human HG-PRTase are positioned in apparently equal hydrogen bonding interactions with the exocyclic N2 of guanine. They are presumably contributing equally to guanine binding in these two active sites and were thus not included for mutational analysis in the present investigation.
The Role of Tyr-127-Sequence alignments among different purine PRTases show a highly conserved domain, commonly known as the PRTase fingerprint domain (Fig. 2). In this conserved sequence, GPRTase from G. lamblia has a Tyr at position 127, whereas all the other purine PRTases have an Ile at the corresponding position. In the three-dimensional structure of GPRTase, the Tyr-127 residue appears to stack below the guanine ring with its phenolic hydroxyl group placed between the purine exocyclic O6 and Lys-152 (Fig. 1A). A Y127I mutant was generated, purified and characterized. The K m value for guanine was obtained from the individual K m(app) values obtained at various PRPP concentrations by extrapolating to the infinite concentration of PRPP. The K m for guanine in this mutant enzyme-catalyzed reaction was determined to be 2.8 Ϯ 0.2 M and the k cat for GMP synthesis was 4.8 Ϯ 0.1 s Ϫ1 . These values contrast an unusually high K m of 16.4 Ϯ 1.8 M and k cat of 76.7 Ϯ 2.5 s Ϫ1 for the wild-type enzyme. In fact, the kinetic constants of GPRTase Y127I mutant are more in line with those of the other purine PRTases, which universally have Ile at the corresponding position (Table I). The Ile residue allows for improved binding of guanine, probably by replacing the bulky phenyl group of Tyr, which apparently hampers proper positioning of the purine base in the binding pocket. However, the mutation also decreases the k cat value by 16-fold, suggesting a reduced release of GMP from the active site which is most likely the rate-limiting step of the enzyme-catalyzed forward reaction (4). The net result from this mutation thus demonstrates little change in catalytic efficiency (k cat /K m ). In T. foetus HGXPRTase, the conserved Ile-104 was also found to be involved in purine binding (22), where it was observed that the I104G mutant has a 6.5-fold decreased binding affinity to guanine.
The affinity of GMP binding to G. lamblia GPRTase was also monitored using fluorescence quenching. Binding of GMP to the enzyme induced quenching of the intrinsic fluorescence of Trp-180 (Fig. 3), which is stacked on top of the bound guanine moiety (Fig. 1A). The dissociation constants, obtained by titration, were determined to be 36.7 Ϯ 2.1 M for the wild type enzyme, whereas for the Y127I mutant it was 8.0 Ϯ 0.7 M. This 4 -5-fold increased binding affinity of GMP to the mutant enzyme is probably responsible for the lowered k cat of the Y127I mutant catalyzed reaction. In the enzyme-catalyzed forward reaction, the release of GMP as the rate-limiting step has been proposed earlier in the case of human HGPRTase (4) and T. foetus HGXPRTase-catalyzed reactions (22). The significantly higher k cat value of wild type G. lamblia GPRTasecatalyzed forward reaction not only reflects a facilitated release of GMP, but also suggests a primary in vivo function of the enzyme for synthesis of guanine nucleotides in Giardia.
The Role of Leu-186 -Leu-186, as seen in the active site of GPRTase (Fig. 1A), provides some apparent nonpolar interactions with the C2 end of bound guanine moiety. This particular amino acid residue is well conserved among all the purine PRTases with varied purine base specificity (7,11,13). Mutants of Leu-186 in G. lamblia GPRTase were prepared and purified as described and characterized in terms of the kinetics of catalyzed reactions. The K m values for guanine and the k cat values for catalysis in the forward reaction were determined. The kinetic constants listed in Table II indicate that the K m values for guanine for the L186I and the L186V-catalyzed reactions were 26-and 35-fold higher than that for the wildtype enzyme, respectively, resulting in significantly reduced catalytic efficiencies. Thus, slight changes in the side chain length and orientation of Leu-186 seem to have an overwhelmingly negative effect on the affinity of binding to guanine. A drastic shortening of the side chain also appears to have a deleterious effect on guanine binding, as observed in the 22-fold increase of K m for guanine in the L186A mutant-catalyzed reaction. There was, however, relatively little change in the K m for guanine when Leu-186 was replaced with Thr. The L186T mutant has a K m of 18.9 Ϯ 1.9 M as compared with the K m of 16.4 Ϯ 1.8 M for the wild-type, whereas there is a mere 5-fold increase in the K m for guanine for the L186S mutant in comparison to the wild-type enzyme. Thus, a side chain with a hydroxyl group seems to offset the significant increase in K m for guanine observed with other minor changes in the hydrophobic side chain of Leu-186. This discrepancy could be explained by a probable hydrogen bonding between the side chain of Thr or Ser and the exocyclic N2 and heterocyclic N3 of guanine, which would be stronger in the case of Thr compared with Ser at that particular position. Another interesting finding was that the k cat of L186T mutant-catalyzed reaction is only slightly lower than that of the wild type enzyme (43.0 Ϯ 3.6 s Ϫ1 ), thus suggesting a similar K m for GMP as that for the wild type enzyme.
Another interesting observation was the K m of 4.9 Ϯ 0.3 M for guanine in the L186F catalyzed reaction. Considering the drastic decreases in the binding affinity observed even with the slightest changes in the side chain length of Leu-186, the 3-fold increase in binding affinity through a bulky phenyl group substitution in the L186F mutant represents a real surprise. It may reflect the ability of the purine-binding site in GPRTase to accommodate a very large hydrophobic group in assisting purine binding through enhanced hydrophobic interactions. Interestingly, in the structure of T. foetus HGXPRTase (13), there is a Phe-162 at the corresponding site in the purine binding pocket. When Phe-162 was mutated to Leu, neither the specificity nor the affinity of purine base binding changed in the mutant enzyme (22) as compared with the wild type enzyme.
The catalytic efficiencies of these mutants as per their k cat /K m ratios show significant drops with the L186I, L186V,

FIG. 1. Active-site geometry in the purine binding site of G. lamblia GPRTase complexed with immucillin G-PO 4 and PPi (16) (A) and human HGPRTase with GMP (7) (B).
and L186A mutants in comparison to the wild-type catalyzed reaction, whereas there is negligible change in the L186T and the L186F mutants. With the increased binding affinity to guanine, the relatively unchanged catalytic efficiency of L186F can be attributed to the lowered k cat , which may reflect less efficient release of the reaction product GMP.
All the mutants listed in Table II were tested for their ability to accept hypoxanthine and xanthine as substrates, but none of them showed any improvement over the wild-type enzyme in terms of broadened purine substrate specificity (results not shown). Leu-186 is thus apparently not involved in the specificity determination of purine base binding.
Leu-192 of Human HGPRTase-In human HGPRTase, Leu-192 occupies the corresponding position in the purine binding pocket (7) as compared with Leu-186 in G. lamblia GPRTase. Lee et al. (23) characterized a L192T and a L192I mutant of the human HGPRTase and showed its influence on purine binding in terms of the apparent K m values. There was apparently little change in the L192T mutant from the wild type, whereas the apparent K m for guanine in L192I-catalyzed reaction had an increase of about 16-fold, which is very much in agreement with our results from the L186I mutant of GPRTase. In order to characterize and compare Leu-192 in human enzyme more thoroughly with the Leu-186 in GPRTase, mutants of L192 in human HGPRTase were prepared and purified and their kinetic constants determined. As shown in Table III, the L192V, L192S, and L192T mutants show negligible changes in the K m values for guanine, whereas their catalytic efficiencies show drastic decreases over the wild-type enzyme-catalyzed reaction. These human enzyme mutants appear to behave similarly to the corresponding GPRTase mutants, except for the GPRTase L186T mutant, which had a wild-type k cat /K m ratio. An even more contrasting observation was made on the K m for guanine between human L192F mutant and GPRTase L186F mutant. The K m for guanine was determined to be 122.8 Ϯ 25.6 M for the L192F mutant, compared with 3.6 Ϯ 0.5 M for the wild type enzyme, representing a 35-fold increase, whereas the catalytic efficiency decreased also by 160-fold. This is the opposite from what we observed in the GPRTase L186F mutant where the K m for guanine is reduced by 3-fold from 16.4 Ϯ 1.8 M to 4.9 Ϯ 0.3 M (see Table II), and there is little change in the k cat /K m ratio. Thus, the human enzyme with Phe at position 192 appears to have a negative effect on purine binding, whereas the parasite enzyme can accommodate the large Phe side chain and allow for better hydrophobic interaction with the purine base.
The Role of Lys-152-In the crystal structure of GPRTase, Lys-152 was shown to interact with exocyclic O6 over a long distance of 6.3 Å through two ordered water molecules, which would apparently constitute a rather weak interaction (12). When this Lys residue was mutated to Arg in the K152R mutant to shorten this apparent distance (Table IV), there appeared to be no major change in the K m for guanine or the k cat value. Hypoxanthine remained unrecognized as a substrate by the L152R mutant, suggesting that neither the increased side chain length nor the guanidino group of Arg enhanced significantly the binding of 6-oxopurine through interaction with the exocyclic O6.
In T. foetus HGXPRTase, it was shown that the conserved Lys-134, whose ⑀-amino group is within 3 Å of the O6 of 6-oxopurine, is the primary determinant in conferring the specificity of the enzyme toward all three 6-oxopurines: guanine, hypoxanthine, and xanthine. Mutating this Lys to either Ala or Ser in T. foetus HGXPRTase increased the K m for guanine, while  FIG. 3. Fluorescence emission spectra of GPRTase in presence of GMP. Protein in 50 mM Tris-HCl, pH 7.5, 12 mM MgCl 2 , at 2 M concentration was used, at an excitation wavelength of 297 nm, and the emission was monitored from 310 to 400 nm. Fluorescence emission spectra were obtained at different GMP concentrations, and the dissociation constant was determined by the fractional saturation (F) calculated at each ligand concentration. allowing adenine to be accepted as a substrate with an estimated K m in the range of 34 -54 M (22). G. lamblia GPRTase shows no recognition of adenine, hypoxanthine, or xanthine as substrate (5). With the poor binding to the exocyclic O6 by Lys-152, the primary binding sites in GPRTase active site that interact with the purine moiety appear to be Leu-186 with purine C2 and the main-chain carbonyls of Asp-181 and Asp-187 with the exocyclic N2 in guanine via hydrogen bonding. It explains why only guanine is used as substrate. It also suggests that by increasing binding affinity to purine other than interacting with the exocyclic N2 may broaden the substrate specificity beyond guanine. Furthermore, as can be seen in the crystal structure of GPRTase (Fig. 1A), the phenolic hydroxyl of Tyr-127 is placed between the ⑀-amino group of Lys-152 and the purine O6. It probably forms a deterrent to the interaction between them and thus further weakens the power of Lys-152 in dictating 6-oxopurine as substrate. Although the Y127I mutant showed no improvement over the wild type in its binding with hypoxanthine, it nevertheless increases the affinity of guanine binding by 6-fold (Table IV). To verify whether Arg-152 would further strengthen purine binding by the Y127I mutant through a potentially stronger hydrogen bonding with exocyclic O6, a double mutant Y127I/K152R of GPRTase was prepared, purified, and characterized. The K m for guanine was not further lowered from that of the Y127I mutant and was determined to be 2.7 Ϯ 0.3 M (Table IV). The k cat value for the forward reaction remained also relatively unchanged from that by the Y127I mutant and was found to be 8.8 Ϯ 0.4 s Ϫ1 . The dissociation constant for GMP was determined to be 6.8 Ϯ 0.5 M, 5-fold lower than that for the wild-type enzyme (Table V). However, the most dramatic change in the double mutantcatalyzed reaction is that hypoxanthine can be now used as a substrate with an estimated K m value of 54.5 Ϯ 15.5 M and a k cat for the forward reaction of 4.4 Ϯ 0.7 s Ϫ1 (Table IV). Thus, by improving nonspecific 6-oxopurine binding to the active site of GPRTase through stronger hydrophobic interactions via a Y127I mutation and a further strengthening of the hydrogen bonding with the exocyclic O6 by the K152R mutation, the primary dependence of purine binding on hydrogen bonding with Asp-181 and Asp-187 becomes de-emphasized. The substrate specificity becomes relaxed somewhat as a result.
Effect of Enhanced O6 and C2 Bindings-Another attempt was made to further improve the purine binding affinity through a double mutation Y127/L186F. The mutant was prepared and characterized kinetically. The K m for guanine was determined to be 2.8 Ϯ 0.3 M, 6-fold lower than that for the wild-type enzyme. The catalytic efficiency too showed an improvement by 3-fold over the wild-type enzyme catalyzed reaction (Table IV). There was, however, no demonstrable utilization of hypoxanthine as a substrate by this double mutant.
A triple mutant Y127I/K152R/L186F was then prepared. It showed a dramatic improvement in both the K m for guanine and in the catalytic efficiency in utilizing guanine. The 6-fold decrease in the K m for guanine and the 4-fold increase in the   k cat /K m ratio over the wild-type enzyme clearly indicates the complementarity of the enhanced binding obtained by three major changes in the structure of the active site in GPRTase, First, the replacement of Tyr-127 with the more conserved and less intrusive Leu; second, extending the side chain length by the K152R mutation, thus enabling enhanced interaction with the O6 of the purine moiety; third, the increased hydrophobic interactions by the presence of a Phe-186 at the base of the purine ring. The enhanced guanine binding obtained by these site-specific changes also has a supplementary effect on hypoxanthine utilization as a substrate. The triple mutant catalyzes the formation of IMP from hypoxanthine with a K m of 29.8 Ϯ 4.1 M and a k cat of 8.0 Ϯ 0.6 s Ϫ1 (Table IV and Fig. 4) This represents a 2-fold decrease in the K m and a 3.3-fold increase in the catalytic efficiency compared with the Y172I/K152R double mutant (Table IV).
The K d values for GMP binding to the Y127I/L186F and the Y127I/K152R/L186F mutant enzymes were 7.7 Ϯ 0.5 and 6.8 Ϯ 0.6 M, respectively. Thus, both the mutants show increased binding to GMP as compared with the wild-type enzyme, representing about 5-6-fold increase in the binding affinity. These results clearly indicate a requirement for enhanced interactions with O6 of the purine moiety combined with strong hydrophobic binding of the purine ring for a 6-oxopurine other than guanine, such as hypoxanthine, to bind effectively to the active site of GPRTase.
Functional Complementation Analysis with E. coli Strain S609 -In order to verify the effectiveness of the triple mutant of GPRTase in converting hypoxanthine to IMP in an in vivo environment, functional complementation of the E. coli strain S609 purine auxotroph (ara, ⌬pro-gpt-lac, thi, hpt, pup, purH, J, strA) with the triple mutant was performed (20). DNA transformants of E. coli were grown in minimal media supplemented with specific nucleobases. Fig. 5 shows that E. coli S609, transformed with the wild-type GPRTase expression plasmid, could grow in the presence of guanine, but showed very poor growth in the presence of hypoxanthine or without the presence of any purine base. The Y127I plasmid-transformed cells behaved quite similarly to the wild-type GPRTase transfected cells in guanine-supplemented medium. E. coli S609 cells transfected with either the Y127I/K152R or the Y127I/K152R/ L186F plasmid grew also to a similar extent as the wild type GPRTase transfected cells on guanine. However, they also grew when supplemented with hypoxanthine and reached about 50% of the final level of growth when compared with that from growing on guanine. Thus, the ability to phosphoribosylate hypoxanthine by the two mutant enzymes could be demonstrated in transfected cells via complementing the auxotrophy of the E. coli strain S609.
We believe that what we have accomplished here is a major finding in our efforts to try to understand the molecular mechanism underlining the strict guanine specificity of G. lamblia GPRTase. This newly gained knowledge should also enable us to design new, potent and specific inhibitors of G. lamblia GPRTase. Guanine derivatives with an extended exocyclic O6 substituent up to a length of 6.3 Å could be developed into a potent inhibitor of this parasite enzyme while incapable of binding to the host enzyme at all.
Conclusion-The purine-binding site in G. lamblia GPRTase has a unique structural feature, which leads to the unusually restrictive purine substrate specificity of the enzyme. The weak interaction between Lys-152 and the exocyclic O6 of bound 6-oxopurine makes the apparent interaction between Asp-181, Asp-187, and the N2 group of guanine crucial for its binding. Consequently, only guanine can bind to the active site of this enzyme. By strengthening the less specific hydrophobic interactions between the purine ring and residues Leu-186 and Tyr-127, coupled with a stronger hydrogen bonding to the exocyclic O6 of purine by mutating Lys-152 to Arg, hypoxanthine can be used as substrate of the mutant enzyme. The insight gained through our intentional alteration of the purine specificity of this enzyme suggests specific routes for the design of effective anti-parasitic agents targeted to the enzyme.