Steady-state kinetics of the schistosomal hypoxanthine-guanine phosphoribosyltransferase

Abstract

Schistosomiasis is a trematode infection of some 200 million people. The hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) of the major etiologic agent, Schistosoma mansoni, has been proposed as a potential target for antischistosomal chemotherapy [Dovey, H. F., McKerrow, J. H., & Wang, C. C. (1984) Mol. Biochem. Parasitol, 11, 157-167]. The steady-state kinetic mechanism for the schistosomal HGPRTase has been determined by including both hypoxanthine and guanine in the forward and reverse reactions under identical conditions. Double-reciprocal plots of initial velocity versus the concentration of one substrate, at a series of fixed concentrations of the other, give groups of intersecting straight lines indicating a sequential mechanism for the schistosomal HGPRTase-catalyzed reactions. In product inhibition studies, the results show that magnesium pyrophosphate (MgPPi) is a noncompetitive inhibitor with respect to dimagnesium phosphoribose pyrophosphate (Mg2PRPP), hypoxanthine, and guanine. Also, magnesium inosine monophosphate (MgIMP) and magnesium guanosine monophosphate (MgGMP) are noncompetitive inhibitors with respect to hypoxanthine or guanine, respectively, but are competitive inhibitors to Mg2PRPP. Furthermore, Mg2PRPP is a competitive inhibitor with respect to MgIMP and MgGMP but is a non-competitive inhibitor to MgPPi. The minimum kinetic model which fits the experimental data is an ordered bi-bi mechanism, where the substrates bind to the enzyme in a defined order (first Mg2PRPP followed by the purine bases), while products are released in sequence (first MgPPi followed by MgIMP or MgGMP).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential inhibitory effects of GMP‐2′,3′‐dialdehyde on human and schistosomal hypoxanthine–guanine phosphoribosyltransferases

“…2 pM; Giacomello and Salerno, 1978) is threefold higher than that for schistosomal HGPRTase (18.25 1.3 pM for GPRTase and 9.3? 1.1 pM for HPRTase; Yuan et al, 1992). Thus, PRibPP can bind to the latter with a higher affinity, but fail to protect it from ox-GMP binding at a concentration as high as 1.0 rnM (see Fig.…”

“…These elucidations may help to explain why in steadystate kinetic studies of the schistosomal HGPRTase-catalyzed reactions, GMP was a competitive inhibitor of PRibPP and vice versa with K, = 55 +-2 pM and 31 -+ 1 pM, respectively (Yuan et al, 1992). In the human HGPRTase-catalyzed reactions, GMP and PRibPP apparently compete also with each other for binding to the active site (Giacomello and Salerno, 1978).…”

Purine Phosphoribosyltransferases

“…For those enzymes that have been studied, the forward reaction appears to be ordered and sequential with PRPP binding first followed by the purine base (3)(4)(5). After catalysis, pyrophosphate (PP i ) is released before the nucleotide.…”

Inactivation of Tritrichomonas Foetus and Schistosoma Mansoni Purine Phosphoribosyltransferases by Arginine‐Specific Reagents

“…Tosylphenylalanylchloromethane (TosPheCH,Cl)-treated trypsin was from Worthington Biochemical Corp. Acetonitrile used in reversephase HPLC was purchased from Fisher. Agpt-pro-lac, thi, hpt) transformed with the pBSprt expression plasmid (Craig et al, 1991) by the method described previously (Yuan et al, 1992). Recombinant human HGPRT was purified from the same strain of E. coli transformed with pBAcprt expression plasmid by a previously described procedure (Kanaaneh et ai., 1994).…”