TY - JOUR
T1 - Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry
AU - Wang, Fang
AU - Scapin, Giovanna
AU - Blanchard, John S.
AU - Angeletti, Ruth Hogue
PY - 1998/2
Y1 - 1998/2
N2 - 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.
AB - 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.
KW - Amide hydrogen exchange
KW - Diaminopimelate dehydrogenase
KW - Electrospray ionization mass spectrometry
KW - Protein conformational change
KW - Substrate binding
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U2 - 10.1002/pro.5560070208
DO - 10.1002/pro.5560070208
M3 - Article
C2 - 9521104
AN - SCOPUS:0031890650
SN - 0961-8368
VL - 7
SP - 293
EP - 299
JO - Protein Science
JF - Protein Science
IS - 2
ER -