STEADY-STATE KINETICS OF THE HYPOXANTHINE-GUANINE-XANTHINE PHOSPHORIBOSYLTRANSFERASE FROM TRITRICHOMONAS-FETUS - THE ROLE OF THREONINE-47

Tritrichomonas foetus, an anaerobic flagellated protozoan, causes urog enital trichomoniasis in cattle. Hypoxanthine-guanine-xanthine phospho ribosyl transferase (HGXPRTase), an essential enzyme in T. foetus requ ired for salvaging exogenous purine bases, has been regarded as a prom ising target for anti-tritrichomonial chemotherapy. The steady-state k inetic analyses of synthesis and pyrophosphorolysis of IMP, GMP, and X MP and product inhibition studies have been used to elucidate the reac tion mechanisms. Double-reciprocal plots of initial velocities versus the varying concentrations of one substrate at a fixed concentration o f the other show intersecting lines indicating a sequential mechanism for both the forward and the reverse reactions. In terms of the k(cat) /K-m ratios, hypoxanthine is the most effective substrate whereas guan ine and xanthine are converted equally well into their corresponding n ucleotides. The minimum kinetic model from the data in product inhibit ion studies is an ordered bi-bi mechanism, where the substrates bind t o the enzyme (first PRPP followed by the purine bases), and the produc ts released (first PPi followed by purine nucleotide) in a defined ord er. The K(m)s for PPI in the T. foetus HGXPRTase-catalyzed reactions a re unusually high, close to the millimolar range. Since the crystal st ructure of this enzyme [Somoza et al. (1996) Biochemistry 35, 7032-704 0] suggest potential binding between the threonine-47 in a conserved c is-peptide loop and PPi whereas human HGPRTase has lysine-68 [Eads et al, (1994) Cell 78, 325-334] at the corresponding position, we prepare d a T47K enzyme mutant and found in the T47K-catalyzed reaction a 4-10 -fold decrease of K-m for PPi. The lack of ionic interactions between Thr-47 and PPi and an increased distance between the loop and the acti ve site as compared to the human HGPRTase are thus proposed to be resp onsible for the high K-m for PPi in the T. foetus HGXPRTase-catalyzed reaction.