Use of amide exchange mass spectrometry to study conformational changes within the endopolygalacturonase II-homogalacturonan-polygalacturonase inhibiting protein system.
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.
King
Daniel
D
Complex Carbohydrate Research Center, Department of Chemistry, University of Georgia, 220 Riverbend Road, Athens, Georgia 30602-4712, USA.
Bergmann
Carl
C
Orlando
Ron
R
Benen
Jacques A E
JA
Kester
Harry C M
HC
Visser
Jaap
J
D000577:Amides; D001234:Aspergillus niger / Q000201:enzymology; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D013058:Mass Spectrometry / Q000379:methods; D010368:Pectins / Q000737:chemistry* / Q000378:metabolism; D027805:Phaseolus / Q000201:enzymology / Q000382:microbiology; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011096:Polygalacturonase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D000577:Amides; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D046911:Macromolecular Substances; C081479:PGIP protein, plant; D010940:Plant Proteins; D010368:Pectins; C400187:endopolygalacturonase II; D011096:Polygalacturonase; C003181:polygalacturonic acid