Hydrogen exchange/electrospray ionization mass spectrometry studies of substrate and inhibitor binding and conformational changes of Escherichia coli dihydrodipicolinate reductase.

Abstract

Escherichia coli dihydrodipicolinate reductase is one of seven enzymes in the succinylase pathway of bacterial L-lysine biosynthesis. The binding of NADH, a substrate, and 2,6-pyridinedicarboxylate, an inhibitor, to the recombinant, overexpressed enzyme has been analyzed using hydrogen/deuterium exchange and electrospray ionization/mass spectrometry. NADH binding reduces the extent of deuterium exchange, as does the subsequent binding of 2,6-pyridinedicarboxylate. Pepsin digestion of the deuterated enzyme and enzyme-inhibitor complex coupled with liquid chromatography/mass spectrometry has allowed the identification of four peptides whose deuterium exchange slows considerably upon the binding of the substrate or inhibitor. Two of these peptides represent regions known or thought to bind NADH and 2,6-pyridinedicarboxylate. Two additional peptides are located at the interdomain hinge region and are proposed to be exchangeable in the "open", catalytically inactive, conformation but are nonexchangeable in the "closed", catalytically active conformation formed after NADH and 2,6-pyridinedicarboxylate binding and domain closure. These studies provide a clear example of a catalytically essential domain movement in this enzyme.