Mass spectrometry as a novel approach to probe cooperativity in multimericenzymatic systems

Investigating cooperativity in multimeric enzymes is of utmost interest to improve our understanding of the mechanism of enzymatic regulation. In the present article, or e propose a novel approach based on mass spectrometry t o probe cooperativity in the binding of a ligand to a multisubunit enzyme. This approach presents the selective advantage of giving a direct insight i nto all the subsequent Ligation states that are formed in solution as the L igand is added to the enzyme. A quantitative interpretation of the electros pray ionization (ESI) mass spectra gives the relative abundance of all the distinct enzymatic species, which allows one to directly deduce the coopera tivity of the system. The overall method is described for the addition of t he oxidized cofactor nicotinamide adenine dinucleotide (NAD(+d)) to a dimer ic mutant of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydro genase (GPDH). It is then applied to four tetrameric enzymes: sturgeon musc le GPDH, wild type and S48G mutant of GPDH from B. stearother mophilus, and alcohol dehydrogenase (ADH) from Bakers yeast. The results illustrate the possibilities offered by this new technique. First, mass spectrometry allow s a control of the enzymes before the addition of NAD(+). Second, the coope rative behavior can be drawn from one single ESI mass spectrum, which makes the method highly attractive in terms of the amount of biological material required. Above all, the major bene fit Lies in the direct visualization o f all the enzymatic species that are in equilibrium in solution. The direct measurement of cooperativity readily resolve the inconvenience of the clas sical approaches employed in this field, which all need to model the experi mental data in order to get the cooperative behavior of the system. (C) 200 1 Academic Press.