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 antitritrichomonal chemotherapy. Structural data on all the type I PRTases reveal a highly flexible, 11-14-amino acid loop, presumably covering the active site. 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 dynamics of the loop in T. foetus HGXPRTase were investigated using site-directed mutants, stop-flow kinetics, chemical modification, and two-dimensional (1)H-(15)N heteronuclear NMR relaxation experiments. S73A, Y74F, and Y74E mutants of HGXPRTase exhibited a 5-7-fold increase in K(m) for guanine and a 3-5-fold 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 progressed, suggesting movement of the loop during catalysis. In the presence of 9-deazaguanine and PRPP, the rapidly quenched fluorescence was not recovered, suggesting a closed loop form. The accessibility of Trp74 in the flexible loop of the mutant enzyme was also analyzed using N-bromosuccinimide (NBS), which reacts specifically with the tryptophan residue. NBS reacted with the only tryptophan in the Y74W mutant enzyme and rendered the enzyme inactive. GMP or PRPP alone failed to protect the enzyme from NBS inactivation. However, the presence of both 9-deazaguanine and PPRP protected the enzyme, allowing it to retain up to 70% of its activity. An S75H mutant, labeled with [(15)N]histidine, was used in the (1)H-(15)N NMR study. Spectra obtained in the presence of enzyme substrates indicated an apparent stabilization of the loop only in the presence of 9-deazaguanine and PRPP. These experimental results thus clearly demonstrated stabilization of the flexible loop upon binding of both PRPP and guanine and suggested its involvement in enzyme catalysis.
Munagala
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Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
Basus
V J
VJ
Wang
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CC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics; D010754:Phosphoribosyl Pyrophosphate / Q000737:chemistry; D017433:Protein Structure, Secondary / Q000235:genetics; D012694:Serine / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics; D016835:Tritrichomonas foetus / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D014443:Tyrosine; D012694:Serine; D010754:Phosphoribosyl Pyrophosphate; D014364:Tryptophan; D010430:Pentosyltransferases; C083867:hypoxanthine-guanine-xanthine phosphoribosyltransferase; D008274:Magnesium