title: Quantitative evaluation of protein-protein and ligand-protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry.
abstract: A mass spectrometer coupling electrospray ionization with time-of-flight mass spectrometry (ESI-TOFMS) has been used to investigate the oligomeric species of Escherichia coli citrate synthase, and to determine the effect of nicotinamide adenine dinucleotide (NADH), an allosteric inhibitor of this enzyme, on the equilibrium between the oligomeric forms. An equilibrium mixture of dimers (M = 95,770 Da) and hexamers (M = 287,310 Da) was found under the conditions used (KA = 6.9 x 10(10) M-2), and NADH was observed to bind selectively to the hexamer (KD = 1.1 microM), shifting the equilibrium to the latter form. The power of ESI-TOFMS to measure ions of very large mass-to-charge ratio (up to m/z approximately 10,000 in this case) is shown to be a valuable tool for obtaining accurate information about compositions of noncovalent complexes and equilibrium constants. The measured constants agree with those determined by conventional means.
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Ayed
authors.forename: A N
authors.initials: AN
authors.lastname: Krutchinsky
authors.forename: W
authors.initials: W
authors.lastname: Ens
authors.forename: K G
authors.initials: KG
authors.lastname: Standing
authors.forename: H W
authors.initials: HW
authors.lastname: Duckworth
mesh_terms: D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000378:metabolism; D004798:Enzymes / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D013058:Mass Spectrometry; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry*
chemical_list: D004798:Enzymes; D011506:Proteins; D009243:NAD; D002950:Citrate (si)-Synthase