Characterization of specific binding by mass spectrometry: Associations of E. coli citrate synthase with NADH and 2-azidoATP

Type II citrate synthases (CSs), found in gram-negative bacteria such as Escherichia coli, are subject to strong and specific allosteric inhibition by NADH. Crystallographic studies have shown that the NADH binding sites are located at macromolecular contacts, so that tight binding requires the intact hexameric structure. Although other adenylates, such as 5′-AMP and ATP, are known to influence CS function by inhibiting NADH binding, the exact mechanism of inhibition is currently unknown. In the present study, we have used mass spectrometry to study the adenylate-binding properties of CS by ESI and MALDI-based techniques. More specifically, to investigate where adenylates bind, we have used the photoaffinity label, 2-azidoATP, to label E. coli CS under non-denaturing conditions for MS analysis of tryptic peptides. Different MS techniques are used to show that tryptic peptide T17 (AA168–177) of the CS sequence known to contribute to NADH binding, disappears after photolabeling, while a new photoaffinity-labeled peptide, with a mass 520 Da than that of T17, appears instead. Another new peptide, corresponding to photolabeled peptide T16–18 (a combined peptide arising from incomplete digestion of the same region) has also been detected. By CE-ESI-TOF-MS, ESI-Q3, MALDI-TOF and analysis of complex stoichiometry we conclude that ATP/ATP-analogs, and presumably the whole series of adenylates potentiate CS and the tricarboxylic acid cycle through the identified site of NADH regulation.

Figure optionsDownload high-quality image (90 K)Download as PowerPoint slideResearch highlights▶ Results in this paper help to identify the general allosteric NADH binding pocket of the enzyme citrate synthase. ▶ Photolabeling experiments using the NADH analogs 2-azidoATP were done to characterize the allosteric site by a combination of mass spectrometry and separation techniques. ▶ Mass spectrometry results suggest peptide modifications in the area of the pocket previously characterized by X-ray crystallography.