Role of the Flexible Loop of Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Tritrichomonas foetus in Enzyme Catalysis

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• 作者：Narsimha Munagala ; Vladimir J. Basus ; and Ching C. Wang
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：April 10, 2001
• 年：2001
• 卷：40
• 期：14
• 页码：4303 - 4311
• 全文大小：237K
• 年卷期：v.40,no.14(April 10, 2001)
• ISSN：1520-4995

文摘

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), a type I PRTase,from Tritrichomonas foetus, is a potential target for antitritrichomonal chemotherapy. Structural data onall the type I PRTases reveal a highly flexible, 11-14-amino acid loop, presumably covering the activesite. With the exception of a highly conserved Ser-Tyr dipeptide, the other amino acids constituting theloop vary widely among different PRTases. The roles of the conserved Ser73 and Tyr74 residues in theloop and the dynamics of the loop in T. foetus HGXPRTase were investigated using site-directed mutants,stop-flow kinetics, chemical modification, and two-dimensional ¹H-¹⁵N heteronuclear NMR relaxationexperiments. S73A, Y74F, and Y74E mutants of HGXPRTase exhibited a 5-7-fold increase in K_m forguanine and a 3-5-fold increase in K_m for PRPP compared to that of the wild type, reflecting the decreasedaffinity of binding for the two substrates. The k_cat's for these mutant-catalyzed reactions, however, do notchange appreciably from that of the wild-type enzyme. Stopped-flow fluorescence with a Y74W mutantshowed no apparent quenching by adding either PRPP or GMP alone. When both PRPP and guaninewere added together, however, the fluorescence was rapidly quenched, followed by a slow recovery asthe enzyme-catalyzed reaction progressed, suggesting movement of the loop during catalysis. In the presenceof 9-deazaguanine and PRPP, the rapidly quenched fluorescence was not recovered, suggesting a closedloop form. The accessibility of Trp74 in the flexible loop of the mutant enzyme was also analyzed usingN-bromosuccinimide (NBS), which reacts specifically with the tryptophan residue. NBS reacted with theonly tryptophan in the Y74W mutant enzyme and rendered the enzyme inactive. GMP or PRPP alonefailed to protect the enzyme from NBS inactivation. However, the presence of both 9-deazaguanine andPPRP protected the enzyme, allowing it to retain up to 70% of its activity. An S75H mutant, labeled with[¹⁵N]histidine, was used in the ¹H-¹⁵N NMR study. Spectra obtained in the presence of enzyme substratesindicated an apparent stabilization of the loop only in the presence of 9-deazaguanine and PRPP. Theseexperimental results thus clearly demonstrated stabilization of the flexible loop upon binding of bothPRPP and guanine and suggested its involvement in enzyme catalysis.