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 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.
Lee
C C
CC
Laboratory of Molecular Parasitology and Drug Design, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7360, USA.
Medrano
F J
FJ
Craig
S P
SP
Eakin
A E
AE
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D011756:Diphosphates / Q000378:metabolism; D017353:Gene Deletion; D006147:Guanine / Q000378:metabolism; D019271:Hypoxanthine / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007291:Inosine Monophosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology*
D011756:Diphosphates; D007291:Inosine Monophosphate; D019271:Hypoxanthine; D006147:Guanine; D010754:Phosphoribosyl Pyrophosphate; D007041:Hypoxanthine Phosphoribosyltransferase