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
. 2000 Sep;9(9):1660–1668. doi: 10.1110/ps.9.9.1660

Immucillin-H binding to purine nucleoside phosphorylase reduces dynamic solvent exchange.

F Wang 1, R W Miles 1, G Kicska 1, E Nieves 1, V L Schramm 1, R H Angeletti 1
PMCID: PMC2144693  PMID: 11045613

Abstract

The rate and extent of hydrogen/deuterium (H/D) exchange into purine nucleoside phosphorylase (PNP) was monitored by electrospray ionization mass spectrometry (ESI-MS) to probe protein conformational and dynamic changes induced by a substrate analogue, products, and a transition state analogue. The genetic deficiency of PNP in humans is associated with severe T-cell immunodeficiency, while B-cell immunity remains functional. Inhibitors of PNP have been proposed for treatment of T-cell leukemia, to suppress the graft-vs.-host response, or to counter type IV autoimmune diseases without destroying humoral immunity. Calf spleen PNP is a homotrimer of polypeptide chains with 284 amino residues, molecular weight 31,541. Immucillin-H inhibits PNP with a Kd of 23 pM when only one of the three catalytic sites is occupied. Deuterium exchange occurs at 167 slow-exchange sites in 2 h when no catalytic site ligands are present. The substrate analogue and product prevented H/D exchange at 10 of the sites. Immucillin-H protected 32 protons from exchange at full saturation. When one of the three subunits of the homotrimer is filled with immucillin-H, and 27 protons are protected from exchange in all three subunits. Deuterium incorporation in peptides from residues 132-152 decreased in all complexes of PNP. The rate and/or extent of deuterium incorporation in peptides from residues 29-49, 50-70, 81-98, and 112-124 decreased only in the complex with the transition state analogue. The peptide-specific H/D exchange demonstrates that (1) the enzyme is most compact in the complex with immucillin-H, and (2) filling a single catalytic site of the trimer reduces H/D exchange in the same peptides in adjacent subunits. The peptides most highly influenced by the inhibitor surround the catalytic site, providing evidence for reduced protein dynamic motion caused by the transition state analogue.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Biemann K. Contributions of mass spectrometry to peptide and protein structure. Biomed Environ Mass Spectrom. 1988 Oct;16(1-12):99–111. doi: 10.1002/bms.1200160119. [DOI] [PubMed] [Google Scholar]
  2. Erion M. D., Stoeckler J. D., Guida W. C., Walter R. L., Ealick S. E. Purine nucleoside phosphorylase. 2. Catalytic mechanism. Biochemistry. 1997 Sep 30;36(39):11735–11748. doi: 10.1021/bi961970v. [DOI] [PubMed] [Google Scholar]
  3. Furneaux R. H., Schramm V. L., Tyler P. C. Transition state analogue inhibitors of protozoan nucleoside hydrolases. Bioorg Med Chem. 1999 Nov;7(11):2599–2606. doi: 10.1016/s0968-0896(99)00210-2. [DOI] [PubMed] [Google Scholar]
  4. Johnson R. S., Walsh K. A. Mass spectrometric measurement of protein amide hydrogen exchange rates of apo- and holo-myoglobin. Protein Sci. 1994 Dec;3(12):2411–2418. doi: 10.1002/pro.5560031224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kline P. C., Schramm V. L. Purine nucleoside phosphorylase. Catalytic mechanism and transition-state analysis of the arsenolysis reaction. Biochemistry. 1993 Dec 7;32(48):13212–13219. doi: 10.1021/bi00211a033. [DOI] [PubMed] [Google Scholar]
  6. Kline P. C., Schramm V. L. Purine nucleoside phosphorylase. Inosine hydrolysis, tight binding of the hypoxanthine intermediate, and third-the-sites reactivity. Biochemistry. 1992 Jul 7;31(26):5964–5973. doi: 10.1021/bi00141a003. [DOI] [PubMed] [Google Scholar]
  7. Koellner G., Luić M., Shugar D., Saenger W., Bzowska A. Crystal structure of calf spleen purine nucleoside phosphorylase in a complex with hypoxanthine at 2.15 A resolution. J Mol Biol. 1997 Jan 17;265(2):202–216. doi: 10.1006/jmbi.1996.0730. [DOI] [PubMed] [Google Scholar]
  8. Mandell J. G., Falick A. M., Komives E. A. Identification of protein-protein interfaces by decreased amide proton solvent accessibility. Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14705–14710. doi: 10.1073/pnas.95.25.14705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mandell J. G., Falick A. M., Komives E. A. Measurement of amide hydrogen exchange by MALDI-TOF mass spectrometry. Anal Chem. 1998 Oct 1;70(19):3987–3995. doi: 10.1021/ac980553g. [DOI] [PubMed] [Google Scholar]
  10. Mao C., Cook W. J., Zhou M., Federov A. A., Almo S. C., Ealick S. E. Calf spleen purine nucleoside phosphorylase complexed with substrates and substrate analogues. Biochemistry. 1998 May 19;37(20):7135–7146. doi: 10.1021/bi9723919. [DOI] [PubMed] [Google Scholar]
  11. Miles R. W., Tyler P. C., Furneaux R. H., Bagdassarian C. K., Schramm V. L. One-third-the-sites transition-state inhibitors for purine nucleoside phosphorylase. Biochemistry. 1998 Jun 16;37(24):8615–8621. doi: 10.1021/bi980658d. [DOI] [PubMed] [Google Scholar]
  12. Morrison J. F., Walsh C. T. The behavior and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol. 1988;61:201–301. doi: 10.1002/9780470123072.ch5. [DOI] [PubMed] [Google Scholar]
  13. Schramm V. L. Comparison of initial velocity and binding data for allosteric adenosine monophosphate nucleosidase. J Biol Chem. 1976 Jun 10;251(11):3417–3424. [PubMed] [Google Scholar]
  14. Smith D. L., Deng Y., Zhang Z. Probing the non-covalent structure of proteins by amide hydrogen exchange and mass spectrometry. J Mass Spectrom. 1997 Feb;32(2):135–146. doi: 10.1002/(SICI)1096-9888(199702)32:2<135::AID-JMS486>3.0.CO;2-M. [DOI] [PubMed] [Google Scholar]
  15. Wang F., Blanchard J. S., Tang X. J. Hydrogen exchange/electrospray ionization mass spectrometry studies of substrate and inhibitor binding and conformational changes of Escherichia coli dihydrodipicolinate reductase. Biochemistry. 1997 Apr 1;36(13):3755–3759. doi: 10.1021/bi963065g. [DOI] [PubMed] [Google Scholar]
  16. Wang F., Li W., Emmett M. R., Hendrickson C. L., Marshall A. G., Zhang Y. L., Wu L., Zhang Z. Y. Conformational and dynamic changes of Yersinia protein tyrosine phosphatase induced by ligand binding and active site mutation and revealed by H/D exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Biochemistry. 1998 Nov 3;37(44):15289–15299. doi: 10.1021/bi981481q. [DOI] [PubMed] [Google Scholar]
  17. Wang F., Li W., Emmett M. R., Marshall A. G., Corson D., Sykes B. D. Fourier transform ion cyclotron resonance mass spectrometric detection of small Ca(2+)-induced conformational changes in the regulatory domain of human cardiac troponin C. J Am Soc Mass Spectrom. 1999 Aug;10(8):703–710. doi: 10.1016/S1044-0305(99)00039-2. [DOI] [PubMed] [Google Scholar]
  18. Wang F., Scapin G., Blanchard J. S., Angeletti R. H. Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry. Protein Sci. 1998 Feb;7(2):293–299. doi: 10.1002/pro.5560070208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Zhang Z., Li W., Logan T. M., Li M., Marshall A. G. Human recombinant [C22A] FK506-binding protein amide hydrogen exchange rates from mass spectrometry match and extend those from NMR. Protein Sci. 1997 Oct;6(10):2203–2217. doi: 10.1002/pro.5560061015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Zhang Z., Post C. B., Smith D. L. Amide hydrogen exchange determined by mass spectrometry: application to rabbit muscle aldolase. Biochemistry. 1996 Jan 23;35(3):779–791. doi: 10.1021/bi952227q. [DOI] [PubMed] [Google Scholar]
  21. Zhang Z., Smith D. L. Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation. Protein Sci. 1993 Apr;2(4):522–531. doi: 10.1002/pro.5560020404. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES