Steady-State Kinetics of the Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Tritrichomonas foetus:  The Role of Threonine-47

Abstract

Tritrichomonas foetus, an anaerobic flagellated protozoan, causes urogenital trichomoniasis in cattle. Hypoxanthine-guanine-xanthine phosphoribosyl transferase (HGXPRTase), an essential enzyme in T. foetus required for salvaging exogenous purine bases, has been regarded as a promising target for anti-tritrichomonial chemotherapy. The steady-state kinetic analyses of synthesis and pyrophosphorolysis of IMP, GMP, and XMP and product inhibition studies have been used to elucidate the reaction mechanisms. Double-reciprocal plots of initial velocities versus the varying concentrations of one substrate at a fixed concentration of the other show intersecting lines indicating a sequential mechanism for both the forward and the reverse reactions. In terms of the kcat/Km ratios, hypoxanthine is the most effective substrate whereas guanine and xanthine are converted equally well into their corresponding nucleotides. The minimum kinetic model from the data in product inhibition studies is an ordered bi-bi mechanism, where the substrates bind to the enzyme (first PRPP followed by the purine bases), and the products released (first PPi followed by purine nucleotide) in a defined order. The Kms for PPi in the T. foetus HGXPRTase-catalyzed reactions are unusually high, close to the millimolar range. Since the crystal structure of this enzyme [Somoza et al. (1996) Biochemistry 35, 7032-7040] suggests potential binding between the threonine-47 in a conserved cis-peptide loop and PPi whereas human HGPRTase has lysine-68 [Eads et al. (1994) Cell 78, 325-334] at the corresponding position, we prepared a T47K enzyme mutant and found in the T47K-catalyzed reaction a 4-10-fold decrease of Km for PPi. The lack of ionic interactions between Thr-47 and PPi and an increased distance between the loop and the active site as compared to the human HGPRTase are thus proposed to be responsible for the high Km for PPi in the T. foetus HGXPRTase-catalyzed reaction.

First Crystal Structures of Mycobacterium tuberculosis 6-Oxopurine Phosphoribosyltransferase: Complexes with GMP and Pyrophosphate and with Acyclic Nucleoside Phosphonates Whose Prodrugs Have Antituberculosis Activity

“…17 This appears to be a unique property of MtHGPRT as the reported K m values for other 6-oxopurine PRTases for PRib-PP is mainly independent of the second substrate. 7,8,15,18 One possible explanation for this phenomenon is that PRib-PP could bind in a slightly different orientation depending on the identity of the base. The K m for PRib-PP is also significantly higher than for human 6-oxopurine PRTases (65 μM, guanine as base) demonstrating another unique property of this enzyme.…”

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.…”

The Adenine Phosphoribosyltransferase from Giardia lamblia Has a Unique Reaction Mechanism and Unusual Substrate Binding Properties

“…This is in contrast to all other known purine phosphoribosyltransferases, for which the reaction mechanisms have been determined to be ordered Bi Bi with PRPP binding to the free enzyme first, followed by the purine base, whereas the products are released in the order of PP i first and the purine nucleotide second (10,21,23,24). Binding of PRPP and adenine to the free APRTase was also demonstrated by their fluorescence enhancing and quenching effects on the F25W mutant enzyme.…”

“…Without an exception, PRPP is always bound to the free enzyme first, followed by the binding of the purine base, whereas PP i is released prior to the release of purine nucleotide upon completion of the reaction (21)(22)(23)(24). The OPRTase-catalyzed reaction follows, however, a random Bi Bi mechanism (26).…”