Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 May;8(5):1023–1031. doi: 10.1110/ps.8.5.1023

Ternary complex structure of human HGPRTase, PRPP, Mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding.

G K Balendiran 1, J A Molina 1, Y Xu 1, J Torres-Martinez 1, R Stevens 1, P J Focia 1, A E Eakin 1, J C Sacchettini 1, S P Craig 3rd 1
PMCID: PMC2144341  PMID: 10338013

Abstract

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one- to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (k(cat)/Km) of the forward and reverse reactions were more severely reduced (6- to 30-fold), and the mutant enzyme showed positive cooperativity in binding of alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo - Fc)phi(calc)] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.

Full Text

The Full Text of this article is available as a PDF (479.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bhatia M. B., Vinitsky A., Grubmeyer C. Kinetic mechanism of orotate phosphoribosyltransferase from Salmonella typhimurium. Biochemistry. 1990 Nov 20;29(46):10480–10487. doi: 10.1021/bi00498a009. [DOI] [PubMed] [Google Scholar]
  2. Brünger A. T., Krukowski A., Erickson J. W. Slow-cooling protocols for crystallographic refinement by simulated annealing. Acta Crystallogr A. 1990 Jul 1;46(Pt 7):585–593. doi: 10.1107/s0108767390002355. [DOI] [PubMed] [Google Scholar]
  3. Cleland W. W. Statistical analysis of enzyme kinetic data. Methods Enzymol. 1979;63:103–138. doi: 10.1016/0076-6879(79)63008-2. [DOI] [PubMed] [Google Scholar]
  4. Craig S. P., 3rd, Focia P. J., Fletterick R. J. Substitution of lysine for arginine at position 199 of a hypoxanthine phosphoribosyltransferase interferes with binding of the primary substrate to the active site. Biochim Biophys Acta. 1997 Apr 25;1339(1):1–3. doi: 10.1016/s0167-4838(97)00037-x. [DOI] [PubMed] [Google Scholar]
  5. Eads J. C., Scapin G., Xu Y., Grubmeyer C., Sacchettini J. C. The crystal structure of human hypoxanthine-guanine phosphoribosyltransferase with bound GMP. Cell. 1994 Jul 29;78(2):325–334. doi: 10.1016/0092-8674(94)90301-8. [DOI] [PubMed] [Google Scholar]
  6. Eakin A. E., Guerra A., Focia P. J., Torres-Martinez J., Craig S. P., 3rd Hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi as a target for structure-based inhibitor design: crystallization and inhibition studies with purine analogs. Antimicrob Agents Chemother. 1997 Aug;41(8):1686–1692. doi: 10.1128/aac.41.8.1686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Evans S. V. SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. J Mol Graph. 1993 Jun;11(2):134-8, 127-8. doi: 10.1016/0263-7855(93)87009-t. [DOI] [PubMed] [Google Scholar]
  8. Focia P. J., Craig S. P., 3rd, Eakin A. E. Approaching the transition state in the crystal structure of a phosphoribosyltransferase. Biochemistry. 1998 Dec 8;37(49):17120–17127. doi: 10.1021/bi9821465. [DOI] [PubMed] [Google Scholar]
  9. Focia P. J., Craig S. P., 3rd, Nieves-Alicea R., Fletterick R. J., Eakin A. E. A 1.4 A crystal structure for the hypoxanthine phosphoribosyltransferase of Trypanosoma cruzi. Biochemistry. 1998 Oct 27;37(43):15066–15075. doi: 10.1021/bi981052s. [DOI] [PubMed] [Google Scholar]
  10. Giacomello A., Salerno C. Human hypoxanthine-guanine phosphoribosyltransferase. Steady state kinetics of the forward and reverse reactions. J Biol Chem. 1978 Sep 10;253(17):6038–6044. [PubMed] [Google Scholar]
  11. Henriksen A., Aghajari N., Jensen K. F., Gajhede M. A flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase. Biochemistry. 1996 Mar 26;35(12):3803–3809. doi: 10.1021/bi952226y. [DOI] [PubMed] [Google Scholar]
  12. Hove-Jensen B., Harlow K. W., King C. J., Switzer R. L. Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene. J Biol Chem. 1986 May 25;261(15):6765–6771. [PubMed] [Google Scholar]
  13. Jochimsen B., Nygaard P., Vestergaard T. Location on the chromosome of Escherichia coli of genes governing purine metabolism. Adenosine deaminase (add), guanosine kinase (gsk) and hypoxanthine phosphoribosyltransferase (hpt). Mol Gen Genet. 1975 Dec 30;143(1):85–91. doi: 10.1007/BF00269424. [DOI] [PubMed] [Google Scholar]
  14. Johnson K. A. Rapid quench kinetic analysis of polymerases, adenosinetriphosphatases, and enzyme intermediates. Methods Enzymol. 1995;249:38–61. doi: 10.1016/0076-6879(95)49030-2. [DOI] [PubMed] [Google Scholar]
  15. Kelley W. N., Greene M. L., Rosenbloom F. M., Henderson J. F., Seegmiller J. E. Hypoxanthine-guanine phosphoribosyltransferase deficiency in gout. Ann Intern Med. 1969 Jan;70(1):155–206. doi: 10.7326/0003-4819-70-1-155. [DOI] [PubMed] [Google Scholar]
  16. Krahn J. M., Kim J. H., Burns M. R., Parry R. J., Zalkin H., Smith J. L. Coupled formation of an amidotransferase interdomain ammonia channel and a phosphoribosyltransferase active site. Biochemistry. 1997 Sep 16;36(37):11061–11068. doi: 10.1021/bi9714114. [DOI] [PubMed] [Google Scholar]
  17. LESCH M., NYHAN W. L. A FAMILIAL DISORDER OF URIC ACID METABOLISM AND CENTRAL NERVOUS SYSTEM FUNCTION. Am J Med. 1964 Apr;36:561–570. doi: 10.1016/0002-9343(64)90104-4. [DOI] [PubMed] [Google Scholar]
  18. Munagala N. R., Chin M. S., Wang C. C. Steady-state kinetics of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus: the role of threonine-47. Biochemistry. 1998 Mar 24;37(12):4045–4051. doi: 10.1021/bi972515h. [DOI] [PubMed] [Google Scholar]
  19. Musick W. D. Structural features of the phosphoribosyltransferases and their relationship to the human deficiency disorders of purine and pyrimidine metabolism. CRC Crit Rev Biochem. 1981;11(1):1–34. doi: 10.3109/10409238109108698. [DOI] [PubMed] [Google Scholar]
  20. Ozturk D. H., Dorfman R. H., Scapin G., Sacchettini J. C., Grubmeyer C. Locations and functional roles of conserved lysine residues in Salmonella typhimurium orotate phosphoribosyltransferase. Biochemistry. 1995 Aug 29;34(34):10755–10763. doi: 10.1021/bi00034a007. [DOI] [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Scapin G., Grubmeyer C., Sacchettini J. C. Crystal structure of orotate phosphoribosyltransferase. Biochemistry. 1994 Feb 15;33(6):1287–1294. doi: 10.1021/bi00172a001. [DOI] [PubMed] [Google Scholar]
  23. Scapin G., Ozturk D. H., Grubmeyer C., Sacchettini J. C. The crystal structure of the orotate phosphoribosyltransferase complexed with orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate. Biochemistry. 1995 Aug 29;34(34):10744–10754. doi: 10.1021/bi00034a006. [DOI] [PubMed] [Google Scholar]
  24. Seegmiller J. E., Rosenbloom F. M., Kelley W. N. Enzyme defect associated with a sex-linked human neurological disorder and excessive purine synthesis. Science. 1967 Mar 31;155(3770):1682–1684. doi: 10.1126/science.155.3770.1682. [DOI] [PubMed] [Google Scholar]
  25. Somoza J. R., Chin M. S., Focia P. J., Wang C. C., Fletterick R. J. Crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas foetus. Biochemistry. 1996 Jun 4;35(22):7032–7040. doi: 10.1021/bi953072p. [DOI] [PubMed] [Google Scholar]
  26. Strauss M., Behlke J., Goerl M. Evidence against the existence of real isozymes of hypoxanthine phosphoribosyltransferase. Eur J Biochem. 1978 Sep 15;90(1):89–97. doi: 10.1111/j.1432-1033.1978.tb12578.x. [DOI] [PubMed] [Google Scholar]
  27. Vos S., Parry R. J., Burns M. R., de Jersey J., Martin J. L. Structures of free and complexed forms of Escherichia coli xanthine-guanine phosphoribosyltransferase. J Mol Biol. 1998 Oct 2;282(4):875–889. doi: 10.1006/jmbi.1998.2051. [DOI] [PubMed] [Google Scholar]
  28. Vos S., de Jersey J., Martin J. L. Crystal structure of Escherichia coli xanthine phosphoribosyltransferase. Biochemistry. 1997 Apr 8;36(14):4125–4134. doi: 10.1021/bi962640d. [DOI] [PubMed] [Google Scholar]
  29. Wilson J. M., Young A. B., Kelley W. N. Hypoxanthine-guanine phosphoribosyltransferase deficiency. The molecular basis of the clinical syndromes. N Engl J Med. 1983 Oct 13;309(15):900–910. doi: 10.1056/NEJM198310133091507. [DOI] [PubMed] [Google Scholar]
  30. Xu Y., Eads J., Sacchettini J. C., Grubmeyer C. Kinetic mechanism of human hypoxanthine-guanine phosphoribosyltransferase: rapid phosphoribosyl transfer chemistry. Biochemistry. 1997 Mar 25;36(12):3700–3712. doi: 10.1021/bi9616007. [DOI] [PubMed] [Google Scholar]
  31. Yuan L., Craig S. P., 3rd, McKerrow J. H., Wang C. C. Steady-state kinetics of the schistosomal hypoxanthine-guanine phosphoribosyltransferase. Biochemistry. 1992 Jan 28;31(3):806–810. doi: 10.1021/bi00118a024. [DOI] [PubMed] [Google Scholar]
  32. Yuan L., Craig S. P., McKerrow J. H., Wang C. C. The hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni. Further characterization and gene expression in Escherichia coli. J Biol Chem. 1990 Aug 15;265(23):13528–13532. [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES