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Studies of noncovalent complexes of Cex and its inhibitors by mass spectrometry Tešić, Milica

Abstract

Tandem mass spectrometry (MS/MS) has been used to compare gas phase and solution binding of three small-molecule inhibitors to the wild type and three mutant forms of the catalytic domain of Cex, an enzyme that hydrolyses xylan. The inhibitors, xylobiosyl-deoxynojirimycin, xylobiosyl-isofagomine lactam, and xylobiosyl-isofagomine consist of a common distal xylose linked to different proximal aza-sugars. The three mutant forms of the enzyme: Asn44Ala, Gln87Met and Gln87Tyr, alter the binding interactions between Cex and the distal sugar of each inhibitor. An electrospray ionization (ESI) triple quadrupole MS/MS system is used to measure the internal energies, ΔE[sub int], that must be added to gas phase ions to cause dissociation of the noncovalent enzyme-inhibitor complexes. Collision cross sections of ions of the apo-enzyme and enzyme-inhibitor complexes, which are required for the calculations of ΔE[sub int], have also been measured. The results show that, in the gas phase, enzyme-inhibitor complexes have more compact, folded conformations than the corresponding apo-enzyme ions. With the mutant enzymes the effects of substituting a single residue can be detected. The energies required to dissociate the gas phase complexes follow the same trend as the values of ΔG[sup 0/sub dissociation] in solution. This trend is observed both with different inhibitors, which probe binding to the proximal sugar, and with mutants of Cex, which probe binding to the distal sugar. Thus the gas phase complexes appear to retain much of their solution binding characteristics. Hydrogen/deuterium exchange shows that apo-protein ions from solutions of complexes exchange more hydrogens than ions of enzyme-inhibitor complexes from the same solution, indicating a more open conformation of apo-protein ions.

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