Converting the guanine phosphoribosyltransferase from Giardia lamblia to a hypoxanthine-guanine phosphoribosyltransferase.
Guanine phosphoribosyltransferase from Giardia 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 microm. 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 microm. 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.
Munagala
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N
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
Sarver
A E
AE
Wang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D016829:Giardia lamblia / Q000187:drug effects / Q000201:enzymology*; D005873:Giardiasis / Q000188:drug therapy; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D011744:Pyrimidinones / Q000378:metabolism; D011758:Pyrroles / Q000378:metabolism; D013329:Structure-Activity Relationship
D010710:Phosphates; D011744:Pyrimidinones; D011758:Pyrroles; C113100:immucillin G; D007041:Hypoxanthine Phosphoribosyltransferase; D008274:Magnesium