QUANTITATIVE-EVALUATION OF PROTEIN-PROTEIN AND LIGAND-PROTEIN EQUILIBRIA OF A LARGE ALLOSTERIC ENZYME BY ELECTROSPRAY-IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRY

A mass spectrometer coupling electrospray ionization with time-of-flig ht mass spectrometry (ESI-TOFMS) has been used to investigate the olig omeric 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 fo rms, An equilibrium mixture of dimers (M = 95 770 Pa) and hexamers (M = 287 310 Da) was found under the conditions used (K-A = 6.9 X 10(10) M-2), and NADH was observed to bind selectively to the hexamer (K-D = 1.1 mu M), Shifting the equilibrium to the latter form, The power of E SI-TOFMS to measure ions of very large mass-to-charge ratio (up to m/z similar to 10 000 in this ease) is shown to be a valuable tool for ob taining accurate information about compositions of noncovalent complex es and equilibrium constants, The measured constants agree with those determined by conventional means. (C) 1998 John Wiley & Sons, Ltd.