Point mutations in the guanine phosphoribosyltransferase from Giardia lamblia modulate pyrophosphate binding and enzyme catalysis

Guanine phosphoribosyltransferase (GPRTase) from Giardia lamblia, an enzyme required for guanine salvage and necessary for the survival of this parasi tic protozoan, has been kinetically characterized. Phosphoribosyltransfer p roceeds through an ordered sequential mechanism common to many related puri ne phosphoribosyltransferases (PRTases) with alpha-D-5-phosphoribosyl-1-pyr ophosphate (PRPP) binding to the enzyme first and guanosine monophosphate ( GMP) dissociating last. The enzyme is a highly unique purine PRTase, recogn izing only guanine as its purine substrate (K-m = 16.4 mu M) but not hypoxa nthine (K-m > 200 mu M) nor xanthine (no reaction). It also catalyzes both the forward (k(cat) = 76.7 s(-1)) and reverse (k(cat) = 5.8 . s(-1)) reacti ons at significantly higher rates than all the other purine PRTases describ ed to date. However, the relative catalytic efficiencies favor the forward reaction, which can be attributed to an unusually high K-m for pyrophosphat e (PPi) (323.9 mu M) in the reverse reaction, comparable only with the high K-m for PPi (165.5 mu M) in Tritrichomonas foetus HGXPRTase-catalyzed reve rse reaction. As the latter case was due to the substitution of threonine f or a highly conserved lysine residue in the PPi-binding loop [Munagala et a l. (1998) Biochemistry 37, 4045-4051], we identified a corresponding threon ine residue in G. lamblia GPRTase at position 70 by sequence alignment, and then generated a T70K mutant of the enzyme. The mutant displays a 6.7-fold lower K-m for PPi with a twofold increase in the K-m for PRPP. Further att empts to improve PPi binding led to the construction of a T70K/A72G double mutant, which displays an even lower K-m of 7.9 mu M for PPi. However, muta tions of the nearby Gly71 to Glu, Arg, or Ala completely inactivate the GPR Tase, suggesting the requirement of flexibility in the putative PPi-binding loop for enzyme catalysis, which is apparently maintained by the glycine r esidue. We have thus tentatively identified the PPi-binding loop in G. lamb lia GPRTase, and attributed the relatively higher catalytic efficiency in t he forward reaction to the unusual loop structure for poor PPi binding in t he reverse reaction.