Hypoxanthine phosphoribosyltransferases (HPRTs) are of biomedical interest because defects in the
enzyme from humans can result in
gouty arthritis or
Lesch-Nyhan syndrome, and in parasites these
enzymes are potential targets for antiparasite
chemotherapy. In HPRTs, a long flexible loop (active site loop II) closes over the active site during the
enzyme catalyzed reaction. Functional roles for this loop have been proposed but have yet to be substantiated. For the present study, seven
amino acids were deleted from loop II of the
HPRT from Trypanosoma cruzi to probe the functional role of this active site loop in catalysis. The mutant
enzyme (Deltaloop II) was expressed in bacteria, purified by affinity chromatography, and kinetic constants were determined for substrates of both forward (
purine salvage) and reverse (pyrophosphorolysis) reactions catalyzed by the
enzyme. Loop II deletion resulted in moderate (0.6-2.7-fold) changes in the Michaelis constants (K(m)s) for substrates other than
pyrophosphate (PP(i)), for which there was a 5.8-fold increase. In contrast, k(cat) values were severely affected by loop deletion, with rates that were 240-840-fold below those for the wild-type
enzyme. Together with previously reported structural data, these results are consistent with active site loop II participating in transition-state stabilization by precise positioning of the substrates for in line nucleophilic attack and in the liberation of PP(i) as a product of the salvage reaction.