Converting the guanine phosphoribosyltransferase from Giardia lamblia to ahypoxanthine-guanine phosphoribosyltransferase

Guanine phosphoribosyltransferase from Giardia lamblia a key enzyme in the purine salvage pathway, is a potential target for anti-giardiasis chemother apy. Recent structural determination of GPRTase (Shi, W., Munagala, N. R., Wang, C. C., Li, C. M, Tyler, P. C,, Furneaux, R I., Grubmeyer, C., Schramm , V. L., and Alno, S. C. (2000) Biochemistry 39, 6781-6790) showed distinct ive features, which could be responsible for its singular guanine specifici ty. Through characterizing specifically designed site-specific mutants of G PRTase, we identified essential moieties in the active site for substrate b inding. Mutating the unusual Tyr-127 of GPRTase to the highly conserved De results in B-fold lower K-m for guanine, A L186F mutation in GPRTase increa sed the affinity toward guanine by 3.3-fold, whereas the corresponding huma n HGPRTase mutant L192F showed a 33-fold increase in K-m for guanine. A dou ble mutant (Y127I/K152R) of GPRTase retained the improved binding of guanin e and also enabled the enzyme to utilize hypoxanthine as a substrate with a K-m, of 54 +/- 15.5 muM A triple mutant (Y127I/K152/L186F) resulted in fur ther increased binding affinity with both guanine and hypoxanthine with the latter showing a lowered K-m of 29.8 +/- 4.1 muM. Dissociation constants m easured 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 .