Investigation of the functional role of active site loop II in a hypoxanthine phosphoribosyltransferase

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 subst antiated. For the present study, seven amino acids were deleted from loop I I of the HPRT from Try-panosoma cruzi to probe the functional role of this active site loop in catalysis. The mutant enzyme (Delta loop II) was expres sed in bacteria, purified by affinity chromatography, and kinetic constants were determined for substrates of both forward (purine salvage) and revers e (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 (PPi), 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 e nzyme. Together with previously reported structural data, these results are consistent with active site loop II participating in transition-state stab ilization by precise positioning of the substrates for in line nucleophilic attack and in the liberation of PPi as a product of the salvage reaction. (C) 2001 Elsevier Science B.V. All rights reserved.