Steady-state kinetics of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi.
The kinetic mechanism for the reaction catalyzed by the hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi was analyzed to determine the feasibility of designing a parasite-specific mechanism-based inhibitor of this enzyme. The results show that the HPRT from T. cruzi follows an essentially ordered bi-bi reaction, and like its human counterpart also likely forms a dead end complex with purine substrates and the product pyrophosphate. Computational fitting of the kinetics data to multiple initial velocity equations gave results that are consistent with the dead end complex arising when the hypoxanthine- or guanine-bound form of the enzyme binds pyrophosphate rather than the phosphoribosylpyrophosphate substrate of the productive forward reaction. Limited proteolytic digestion was employed to provide additional support for formation of the dead end complex and to estimate the K(d) values for substrates of both the forward and reverse reactions. Due to similarities with the kinetic mechanism of the human HPRT, the results reported here for the HPRT from T. cruzi indicate that the design of a mechanism-based inhibitor of the trypanosomal HPRT, that would not also inhibit the human enzyme, may be difficult. However, the results also show that a potent selective inhibitor of the trypanosomal HPRT might be achieved via the design of a bi-substrate type inhibitor that incorporates analogs of moieties for a purine base and pyrophosphate.
Wenck
Mary Anne
MA
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA.
Medrano
Francisco J
FJ
Eakin
Ann E
AE
Craig
Sydney P
SP
D000818:Animals; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D019271:Hypoxanthine / Q000737:chemistry / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D010754:Phosphoribosyl Pyrophosphate / Q000737:chemistry / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
D011756:Diphosphates; D019271:Hypoxanthine; D010754:Phosphoribosyl Pyrophosphate; D007041:Hypoxanthine Phosphoribosyltransferase; D014357:Trypsin