Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry.
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.
Wang
F
F
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Scapin
G
G
Blanchard
J S
JS
Angeletti
R H
RH
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003013:Clostridium / Q000201:enzymology*; D003903:Deuterium; D006859:Hydrogen; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D010446:Peptide Fragments; D011994:Recombinant Proteins; D009249:NADP; D006859:Hydrogen; D003903:Deuterium; D000594:Amino Acid Oxidoreductases; C020032:diaminopimelate dehydrogenase