The purine transferase from Trypanosoma cruzi as a potential target for bisphosphonate-based chemotherapeutic compounds.

We identified and tested bisphosphonates as inhibitors of a protozoan molecular target. Computational modeling studies demonstrated that these compounds are mimics of the natural substrate of the enzyme. The most potent bisphosphonates in vitro are pamidronate and risedronate, which inhibit the purine transferase from Trypanosoma cruzi in the micromolar range.

Steady-state kinetics of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi.

The kinetic mechanism for the reaction catalyzed by the hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi was analyzed to determine the feasibility of designing a parasite-specific mechanism-based inhibitor of this enzyme. The results show that the HPRT from T. cruzi follows an essentially ordered bi-bi reaction, and like its human counterpart also likely forms a dead end complex with purine substrates and the product pyrophosphate. Computational fitting of the kinetics data to multiple initial velocity equations gave results that are consistent with the dead end complex arising when the hypoxanthine- or guanine-bound form of the enzyme binds pyrophosphate rather than the phosphoribosylpyrophosphate substrate of the productive forward reaction. Limited proteolytic digestion was employed to provide additional support for formation of the dead end complex and to estimate the K(d) values for substrates of both the forward and reverse reactions. Due to similarities with the kinetic mechanism of the human HPRT, the results reported here for the HPRT from T. cruzi indicate that the design of a mechanism-based inhibitor of the trypanosomal HPRT, that would not also inhibit the human enzyme, may be difficult. However, the results also show that a potent selective inhibitor of the trypanosomal HPRT might be achieved via the design of a bi-substrate type inhibitor that incorporates analogs of moieties for a purine base and pyrophosphate.

Saturation mutagenesis, complement selection, and steady-state kinetic studies illuminate the roles of invariant residues in active site loop I of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi.

Hypoxanthine phosphoribosyltransferases (HPRTs) are potential drug targets in the treatment of diseases caused by parasites. Also, defects in the human HPRT can result in gouty arthritis or Lesch-Nyhan syndrome. Active site loop I of HPRTs has been implicated in interactions between enzyme subunits that can influence the relative efficiencies of forward and reverse reactions, but the functional roles for invariant loop I residues (analogous with human Leu67 and Gly69) are poorly understood. Herein, saturation mutagenesis, complement selection, and steady-state kinetics were used to investigate the functional roles for Leu67 and Gly69. Seventy clones from a library of mutants were sequenced and more than 30 different mutations, or combinations of mutations, were identified. Several recombinant HPRTs with mutations at positions 67 and/or 69 supported the growth of a bacterial auxotroph on selective media, but only two of the mutants (L67M and G69S) could be recovered in the soluble fraction from bacteria induced to over-express the enzyme. The results of steady-state kinetic studies for L67M are consistent with the side chain of this residue participating in hydrophobic interactions between dimer subunits that are important for the proper positioning of main chain atoms that influence enzyme chemistry and the binding of PRPP, PPi, and hypoxanthine. The results for mutations at position 69 are consistent with only hydrogen or a small polar side chain being tolerated at this site. Kinetic studies of G69S suggest that side chains of residues at position 69 that project into the active site likely interfere with the binding of PRPP and PPi, as well as the positioning of a metal ion that indirectly influences the binding of purine bases and purine moieties of nucleotide substrates.

Interactions at the dimer interface influence the relative efficiencies for purine nucleotide synthesis and pyrophosphorolysis in a phosphoribosyltransferase.

Enzymes that salvage 6-oxopurines, including hypoxanthine phosphoribosyltransferases (HPRTs), are potential targets for drugs in the treatment of diseases caused by protozoan parasites. For this reason, a number of high-resolution X-ray crystal structures of the HPRTs from protozoa have been reported. Although these structures did not reveal why HPRTs need to form dimers for catalysis, they revealed the existence of potentially relevant interactions involving residues in a loop of amino acid residues adjacent to the dimer interface, but the contributions of these interactions to catalysis remained poorly understood. The loop, referred to as active-site loop I, contains an unusual non-proline cis-peptide and is composed of residues that are structurally analogous with Leu67, Lys68, and Gly69 in the human HPRT. Functional analyses of site-directed mutations (K68D, K68E, K68N, K68P, and K68R) in the HPRT from Trypanosoma cruzi, etiologic agent of Chagas' disease, show that the side-chain at position 68 can differentially influence the K(m) values for all four substrates as well as the k(cat) values for both IMP formation and pyrophosphorolysis. Also, the results for the K68P mutant are inconsistent with a cis-trans peptide isomerization-assisted catalytic mechanism. These data, together with the results of structural studies of the K68R mutant, reveal that the side-chain of residue 68 does not participate directly in reaction chemistry, but it strongly influences the relative efficiencies for IMP formation and pyrophosphorolysis, and the prevalence of lysine at position 68 in the HPRT of the majority of eukaryotes is consistent with there being a biological role for nucleotide pyrophosphorolysis.

Functional roles for amino acids in active site loop II of a hypoxanthine phosphoribosyltransferase.

A flexible loop of amino acids (loop II) closes over the active site of hypoxanthine phosphoribosyltransferase (HPRT) as the enzyme approaches the transition state [Biochemistry 37 (1998) 17120]. Formerly, the deletion of much of loop II from the HPRT of Trypanosoma cruzi resulted in a 2-3 order of magnitude reduction in k(cat) values with relatively modest changes in the Michaelis constants for substrates [Biochim. Biophys. Acta 1537 (2001) 63-70]. However, the contributions of individual loop II residues to catalysis remained poorly understood or have been disputed. Herein, saturation mutagenesis was used to generate relatively random sets of mutations in the 12 residues of active site loop II in the HPRT from T. cruzi and steady-state kinetics was used to investigate reactions catalyzed by the mutants. The results of analyses of 18 different mutations in an evolutionarily invariant Ser-Tyr dipeptide are consistent with interactions, between main chain nitrogen atoms of these residues and the O1A atom of phosphoribosylpyrophosphate (PRPP) or pyrophosphate (PPi), being essential for efficient enzyme chemistry. The results of analyses of 55 mutations in the nine other amino acids in loop II are inconsistent with these residues participating directly in enzyme chemistry, but are consistent with several of their side chains influencing loop flexibility and folding, as well as the efficiency for nucleotide formation relative to pyrophosphorolysis.

Analysis of 6-(2,2-Dichloroacetamido)chrysene interaction with the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi.

Selective inhibition is needed for drugs targeting the hypoxanthine phosphoribosyltransferase of Trypanosoma cruzi, etiologic agent of Chagas' disease. 6-(2,2-Dichloroacetamido)chrysene, was shown herein to be a selective inhibitor of the trypanosomal enzyme. SAR analysis revealed that the 6-amido moiety was essential, but the dichloroaceto moiety was not essential for achieving the low K(i) for this inhibitor. Understanding the molecular basis for these interactions could facilitate the design of selective inhibitors without a chrysene moiety.