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. 1998 Feb;7(2):293–299. doi: 10.1002/pro.5560070208

Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry.

F Wang 1, G Scapin 1, J S Blanchard 1, R H Angeletti 1
PMCID: PMC2143904  PMID: 9521104

Abstract

C. glutamicum meso-diaminopimelate dehydrogenase is an enzyme of the L-lysine biosynthetic pathway in bacteria. The binding of NADPH and diaminopimelate to the recombinant, overexpressed enzyme has been analyzed using hydrogen/deuterium exchange and electrospray ionization/mass spectrometry. NADPH binding reduces the extent of deuterium exchange, as does the binding of diaminopimelate. Pepsin digestion of the deuterated enzyme and enzyme-substrate complexes coupled with liquid chromatography/mass spectrometry have allowed the identification of eight peptides whose deuterium exchange slows considerably upon the binding of the substrates. These peptides represent regions known or thought to bind NADPH and diaminopimelate. One of these peptides is located at the interdomain hinge region and is proposed to be exchangeable in the "open," catalytically inactive, conformation but nonexchangeable in the "closed," catalytically active conformation formed after NADPH and diaminopimelate binding and domain closure. Furthermore, the dimerization region has been localized by this method, and this study provides an example of detecting protein-protein interface regions using hydrogen/deuterium exchange and electrospray ionization.

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Selected References

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  1. Brunhuber N. M., Blanchard J. S. The biochemistry and enzymology of amino acid dehydrogenases. Crit Rev Biochem Mol Biol. 1994;29(6):415–467. doi: 10.3109/10409239409083486. [DOI] [PubMed] [Google Scholar]
  2. Englander J. J., Calhoun D. B., Englander S. W. Measurement and calibration of peptide group hydrogen-deuterium exchange by ultraviolet spectrophotometry. Anal Biochem. 1979 Jan 15;92(2):517–524. doi: 10.1016/0003-2697(79)90693-6. [DOI] [PubMed] [Google Scholar]
  3. Englander J. J., Rogero J. R., Englander S. W. Protein hydrogen exchange studied by the fragment separation method. Anal Biochem. 1985 May 15;147(1):234–244. doi: 10.1016/0003-2697(85)90033-8. [DOI] [PMC free article] [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. Mallikarachchi D., Burz D. S., Allewell N. M. Effects of ATP and CTP on the conformation of the regulatory subunit of Escherichia coli aspartate transcarbamylase in solution: a medium-resolution hydrogen exchange study. Biochemistry. 1989 Jun 27;28(13):5386–5391. doi: 10.1021/bi00439a011. [DOI] [PubMed] [Google Scholar]
  6. Misono H., Soda K. Properties of meso-alpha,epsilon-diaminopimelate D-dehydrogenase from Bacillus sphaericus. J Biol Chem. 1980 Nov 25;255(22):10599–10605. [PubMed] [Google Scholar]
  7. Pavelka M. S., Jr, Jacobs W. R., Jr Biosynthesis of diaminopimelate, the precursor of lysine and a component of peptidoglycan, is an essential function of Mycobacterium smegmatis. J Bacteriol. 1996 Nov;178(22):6496–6507. doi: 10.1128/jb.178.22.6496-6507.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Reddy S. G., Scapin G., Blanchard J. S. Expression, purification, and crystallization of meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum. Proteins. 1996 Aug;25(4):514–516. doi: 10.1002/prot.12. [DOI] [PubMed] [Google Scholar]
  9. Robinson C. V., Gross M., Eyles S. J., Ewbank J. J., Mayhew M., Hartl F. U., Dobson C. M., Radford S. E. Conformation of GroEL-bound alpha-lactalbumin probed by mass spectrometry. Nature. 1994 Dec 15;372(6507):646–651. doi: 10.1038/372646a0. [DOI] [PubMed] [Google Scholar]
  10. Rosa J. J., Richards F. M. An experimental procedure for increasing the structural resolution of chemical hydrogen-exchange measurements on proteins: application to ribonuclease S peptide. J Mol Biol. 1979 Sep 25;133(3):399–416. doi: 10.1016/0022-2836(79)90400-5. [DOI] [PubMed] [Google Scholar]
  11. Scapin G., Reddy S. G., Blanchard J. S. Three-dimensional structure of meso-diaminopimelic acid dehydrogenase from Corynebacterium glutamicum. Biochemistry. 1996 Oct 22;35(42):13540–13551. doi: 10.1021/bi961628i. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Wang F., Tang X. Conformational heterogeneity of stability of apomyoglobin studied by hydrogen/deuterium exchange and electrospray ionization mass spectrometry. Biochemistry. 1996 Apr 2;35(13):4069–4078. doi: 10.1021/bi9521304. [DOI] [PubMed] [Google Scholar]
  15. Woodward C., Simon I., Tüchsen E. Hydrogen exchange and the dynamic structure of proteins. Mol Cell Biochem. 1982 Oct 29;48(3):135–160. doi: 10.1007/BF00421225. [DOI] [PubMed] [Google Scholar]
  16. 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]

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