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 parasitic protozoan, has been kinetically characterized. Phosphoribosyltransfer proceeds through an ordered sequential mechanism common to many related purine phosphoribosyltransferases (PRTases) with α‐D‐5‐phosphoribosyl‐1‐pyrophosphate (PRPP) binding to the enzyme first and guanosine monophosphate (GMP) dissociating last. The enzyme is a highly unique purine PRTase, recognizing only guanine as its purine substrate ( K m  = 16.4 µ m ) but not hypoxanthine ( K m  > 200 µ 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 ) reactions at significantly higher rates than all the other purine PRTases described to date. However, the relative catalytic efficiencies favor the forward reaction, which can be attributed to an unusually high K m for pyrophosphate (PP i ) (323.9 µ m ) in the reverse reaction, comparable only with the high K m for PP i (165.5 µ m ) in Tritrichomonas foetus HGXPRTase‐catalyzed reverse reaction. As the latter case was due to the substitution of threonine for a highly conserved lysine residue in the PP i ‐binding loop [Munagala et al . (1998) Biochemistry 37 , 4045–4051], we identified a corresponding threonine 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 PP i with a twofold increase in the K m for PRPP. Further attempts to improve PP i binding led to the construction of a T70K/A72G double mutant, which displays an even lower K m of 7.9 µ m for PP i . However, mutations of the nearby Gly71 to Glu, Arg, or Ala completely inactivate the GPRTase, suggesting the requirement of flexibility in the putative PP i ‐binding loop for enzyme catalysis, which is apparently maintained by the glycine residue. We have thus tentatively identified the PP i ‐binding loop in G. lamblia GPRTase, and attributed the relatively higher catalytic efficiency in the forward reaction to the unusual loop structure for poor PP i binding in the reverse reaction.