Role of the flexible loop of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus in enzyme catalysis

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), a type I PRTase, from Tritrichomonas foetus, is a potential target for antitr itrichomonal chemotherapy. Structural data on all the type I PRTases reveal a highly flexible, 11-14-amino acid loop, presumably covering the active s ite. With the exception of a highly conserved Ser-Tyr dipeptide, the other amino acids constituting the loop vary widely among different PRTases. The roles of the conserved Ser73 and Tyr74 residues in the loop and the dynamic s of the loop in T. foetus HGXPRTase were investigated using site-directed mutants, stop-flow kinetics, chemical modification, and two-dimensional H-1 -N-15 heteronuclear NMR relaxation experiments. S73A, Y74F, and Y74E mutant s of HGXPRTase exhibited a 5-7-fold increase in K-m for guanine and a 3-5-f old increase in K-m for PRPP compared to that of the wild type, reflecting the decreased affinity of binding for the two substrates. The k(cat)' s for these mutant-catalyzed reactions, however, do not change appreciably from that of the wild-type enzyme. Stopped-flow fluorescence with a Y74W mutant showed no apparent quenching by adding either PRPP or GMP alone. When both PRPP and guanine were added together, however, the fluorescence was rapidly quenched, followed by a slow recovery as the enzyme-catalyzed reaction pro gressed, suggesting movement of the loop during catalysis. In the presence of 9-deazaguanine and PRPP, the rapidly quenched fluorescence was not recov ered, suggesting a closed loop form. The accessibility of Trp74 in the flex ible loop of the mutant enzyme was also analyzed using N-bromosuccinimide ( NBS), which reacts specifically with the tryptophan residue. NBS reacted wi th the only tryptophan in the Y74W mutant enzyme and rendered the enzyme in active. GMP or PRPP alone failed to protect the enzyme from NBS inactivatio n. However, the presence of both 9-deazaguanine and PPRP protected the enzy me, allowing it to retain up to 70% of its activity. An S75H mutant, labele d with [N-15]histidine, was used in the H-1-N-15 NMR study. Spectra obtaine d in the presence of enzyme substrates indicated an apparent stabilization of the loop only in the presence of 9-deazaguanine and PRPP. These experime ntal results thus clearly demonstrated stabilization of the flexible loop u pon binding of both PRPP and guanine and suggested its involvement in enzym e catalysis.