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Use of Amide Exchange Mass Spectrometry To Study Conformational Changes within the Endopolygalacturonase II−Homogalacturonan−Polygalacturonase Inhibiting Protein System

2002; American Chemical Society; Volume: 41; Issue: 32 Linguagem: Inglês

10.1021/bi020119f

1943-295X

Daniel King, Carl Bergmann, Ron Orlando, Jacques Benen, H.C.M. Kester, Jaap Visser,

Phytase and its Applications

Amide exchange mass spectrometry (MS) was used to study the enzyme endopolygalacturonase II (EPG-II) from Aspergillus niger as it binds to an oligosaccharide substrate. A localized decrease in the level of deuterium incorporation in EPG-II of the EPG-II−oligosaccharide complex relative to that of the free EPG-II identified the location of substrate contact, which is in agreement with published site specific mutation studies. In addition, when bound with substrate, regions of EPG-II remote from the substrate binding site became exposed to the solvent, as revealed by an increase in the amount of incorporated deuterium, indicating a conformational change in the enzyme. Fluorescence experiments were performed to provide additional evidence for an altered conformation of EPG-II as a result of substrate binding. This novel application of amide exchange-MS to the study of protein−carbohydrate binding has, for the first time, described in detail the conformational changes associated with EPG-II when it binds a substrate. Amide exchange-MS was also used to study the interactions of EPG-II and the polygalacturonase inhibitor protein (PGIP). Mass spectral data of the EPG-II−oligosaccharide complex in the presence of Phaseolus vulgaris PGIP indicate that the inhibitor contacts EPG-II at a site remote from the substrate binding cleft, and is restricting the conformational changes of EPG-II. Fluorescence experiments also revealed that upon binding of PGIP, the conformational changes mentioned above for the EPG-II−substrate complex are minimized. These results, together with previously reported data, point to a location on EPG-II for interaction with PGIP as well as a possible mechanism for noncompetitive inhibition of EPG-II.