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

Fang Wang, Giovanna Scapin, John S. Blanchard, Ruth Hogue Angeletti

Research output: Contribution to journalArticle

30 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)293-299
Number of pages7
JournalProtein Science
Volume7
Issue number2
StatePublished - Feb 1998

Fingerprint

diaminopimelate dehydrogenase
Clostridium
Deuterium
NADP
Mass spectrometry
Hydrogen
Mass Spectrometry
Electrospray ionization
Substrates
Enzymes
Peptides
Conformations
Dimerization
Electrospray Ionization Mass Spectrometry
Pepsin A
Biosynthetic Pathways
Liquid chromatography
Hinges
Liquid Chromatography
Lysine

Keywords

  • Amide hydrogen exchange
  • Diaminopimelate dehydrogenase
  • Electrospray ionization mass spectrometry
  • Protein conformational change
  • Substrate binding

ASJC Scopus subject areas

  • Biochemistry

Cite this

Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry. / Wang, Fang; Scapin, Giovanna; Blanchard, John S.; Angeletti, Ruth Hogue.

In: Protein Science, Vol. 7, No. 2, 02.1998, p. 293-299.

Research output: Contribution to journalArticle

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