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Quantitative evaluation of protein–protein and ligand–protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry

✍ Scribed by Ayeda Ayed; Andrew N. Krutchinsky; Werner Ens; Kenneth G. Standing; Harry W. Duckworth

Publisher
John Wiley and Sons
Year
1998
Tongue
English
Weight
138 KB
Volume
12
Category
Article
ISSN
0951-4198

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✦ Synopsis

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 (K A = 6.9 Â 10 10 M À2 ), and NADH was observed to bind selectively to the hexamer (K D = 1.1 mM), 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 $ 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.

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