Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1974 Jul;71(7):2833–2837. doi: 10.1073/pnas.71.7.2833

The Active Site of Staphylococcal Nuclease: Paramagnetic Relaxation of Bound Nucleotide Inhibitor Nuclei by Lanthanide Ions

Bruce Furie *, John H Griffin *, Richard J Feldmann †, Edward A Sokoloski ‡, Alan N Schechter *
PMCID: PMC388566  PMID: 4528015

Abstract

The structure of 3′,5′-thymidine diphosphate bound in the active site of staphylococcal nuclease (EC 3.1.4.7) was studied by measuring the relaxation rate enhancement of substrate analog nuclei by a paramagnetic metal ion. The lanthanide ion, Gd(III), was substituted for Ca(II) in the formation of the ternary complex of nuclease-Gd(III)-3′,5′-thymidine diphosphate. Measurements were made of the transverse relaxation rates of protons and the longitudinal and transverse relaxation rates of the phosphorus nuclei of the bound nucleotide. Internuclear distances between the metal ion and atoms of the 3′,5′-thymidine diphosphate nucleotide were determined from these data by the Solomon-Bloembergen equation. In general, these distances corresponded closely to those determined by previous x-ray crystallography of the thymidine diphosphate complex.

These internuclear distances were also used with a computer program and graphics display to solve for metal-nucleotide geometries, which were consistent with the experimental data. A geometry similar to the structure of the metal-nucleotide complex bound to nuclease determined by x-ray analysis was one of the solutions to this computer modeling process. For staphylococcal nuclease, the nuclear magnetic resonance and x-ray methods yield compatible high resolution information about the structure of the active site. However, differences of uncertain significance exist between the two structures.

Keywords: nuclear magnetic resonance; 3′,5′-thymidine diphosphate; gadolinium; calcium-binding proteins; substrate geometry

Full text

PDF
2833

Selected References

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

  1. Arnone A., Bier C. J., Cotton F. A., Day V. W., Hazen E. E., Jr, Richardson D. C., Yonath A., Richardson J. S. A high resolution structure of an inhibitor complex of the extracellular nuclease of Staphylococcus aureus. I. Experimental procedures and chain tracing. J Biol Chem. 1971 Apr 10;246(7):2302–2316. [PubMed] [Google Scholar]
  2. Barry C. D., North A. C., Glasel J. A., Williams R. J., Xavier A. V. Quantitative determination of mononucleotide conformations in solution using lanthanide ion shift and broadenine NMR probes. Nature. 1971 Jul 23;232(5308):236–245. doi: 10.1038/232236a0. [DOI] [PubMed] [Google Scholar]
  3. Bennick A., Campbell I. D., Dwek R. A., Price N. C., Radda G. K., Salmon A. G. Relationship between conformationally sensitive probe binding sites on phosphorylase b. Nat New Biol. 1971 Dec 1;234(48):140–143. doi: 10.1038/newbio234140a0. [DOI] [PubMed] [Google Scholar]
  4. Birnbaum E. R., Gomez J. E., Darnall D. W. Rare earth metal ions as probes of electrostatic binding sites in proteins. J Am Chem Soc. 1970 Aug 26;92(17):5287–5288. doi: 10.1021/ja00720a086. [DOI] [PubMed] [Google Scholar]
  5. Brewer C. F., Sternlicht H., Marcus D. M., Grollman A. P. Binding of 13 C-enriched -methyl-D-glucopyranoside to concanavalin A as studied by carbon magnetic resonance. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1007–1011. doi: 10.1073/pnas.70.4.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Campbell I. D., Dobson C. M., Williams R. J., Xavier A. V. The determination of the structure of proteins in solution: lysozyme. Ann N Y Acad Sci. 1973 Dec 31;222:163–174. doi: 10.1111/j.1749-6632.1973.tb15259.x. [DOI] [PubMed] [Google Scholar]
  7. Cuatrecasas P., Wilchek M., Anfinsen C. B. Selective enzyme purification by affinity chromatography. Proc Natl Acad Sci U S A. 1968 Oct;61(2):636–643. doi: 10.1073/pnas.61.2.636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Darnall D. W., Birnbaum E. R. Rare earth metal ions as probes of calcium ion binding sites in proteins. Neodymium(3) acceleration of the activation of trypsinogen. J Biol Chem. 1970 Dec 10;245(23):6484–6486. [PubMed] [Google Scholar]
  9. Dwek R. A., Richards R. E., Morallee K. G., Nieboer E., Williams R. J., Xavier A. V. The lanthanide cations as probes in biological systems. Proton relaxation enhancement studies for model systems and lysozyme. Eur J Biochem. 1971 Jul 29;21(2):204–209. doi: 10.1111/j.1432-1033.1971.tb01457.x. [DOI] [PubMed] [Google Scholar]
  10. Feldman R. J., Bacon C. R., Cohen J. S. Versatile interactive graphics display system for molecular modelling by computer. Nature. 1973 Jul 13;244(5411):113–115. doi: 10.1038/244113a0. [DOI] [PubMed] [Google Scholar]
  11. Levitzki A., Reuben J. Abortive complexes of -amylases with lanthanides. Biochemistry. 1973 Jan 2;12(1):41–44. doi: 10.1021/bi00725a007. [DOI] [PubMed] [Google Scholar]
  12. Matthews B. W., Weaver L. H. Binding of lanthanide ions to thermolysin. Biochemistry. 1974 Apr 9;13(8):1719–1725. doi: 10.1021/bi00705a025. [DOI] [PubMed] [Google Scholar]
  13. Mildvan A. S., Cohn M. Aspects of enzyme mechanisms studies by nuclear spin relazation induced by paramagnetic probes. Adv Enzymol Relat Areas Mol Biol. 1970;33:1–70. doi: 10.1002/9780470122785.ch1. [DOI] [PubMed] [Google Scholar]
  14. Morávek L., Anfinsen C. B., Cone J. L., Taniuchi H. The large scale preparation of an extracellular nuclease of Staphylococcus aureus. J Biol Chem. 1969 Jan 25;244(2):497–499. [PubMed] [Google Scholar]
  15. Nowak T., Mildvan A. S., Kenyon G. L. Nuclear relaxation and kinetic studies of the role of Mn 2+ in the mechanism of enolase. Biochemistry. 1973 Apr 24;12(9):1690–1701. doi: 10.1021/bi00733a005. [DOI] [PubMed] [Google Scholar]
  16. Reuben J. Gadolinium (3) as a paramagnetic probe for proton relaxation studies of biological macromolecules. Binding to bovine serum albumin. Biochemistry. 1971 Jul 20;10(15):2834–2838. doi: 10.1021/bi00791a005. [DOI] [PubMed] [Google Scholar]
  17. Sloan D. L., Mildvan A. S. Magnetic resonance studies of the geometry of bound nicotinamide adenine dinucleotide and isobutyramide on spin-labeled alcohol dehydrogenase. Biochemistry. 1974 Apr 9;13(8):1711–1718. doi: 10.1021/bi00705a024. [DOI] [PubMed] [Google Scholar]
  18. Sykes B. D., Schmidt P. G., Stark G. R. Aspartate transcarbamylase. A study by transient nuclear magnetic resonance of the binding of succinate to the native enzyme and its catalytic subunit. J Biol Chem. 1970 Mar 10;245(5):1180–1189. [PubMed] [Google Scholar]
  19. Ts'o P. O., Schweizer M. P., Hollis D. P. Contribution of nuclear magnetic resonance to the study of the structure and electronic aspects of nucleic acids. Ann N Y Acad Sci. 1969 May 16;158(1):256–297. doi: 10.1111/j.1749-6632.1969.tb56226.x. [DOI] [PubMed] [Google Scholar]
  20. Villafranca J. J., Mildvan A. S. The mechanism of aconitase action. 3. Detection and properties of enzyme-metal-substrate and enzyme-metal-inhibitor bridge complexes with manganese(II) and iron(II). J Biol Chem. 1972 Jun 10;247(11):3454–3463. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES