title: Kinetic and substrate binding analysis of phosphorylase b via electrospray ionization mass spectrometry: a model for chemical proteomics of sugar phosphorylases.
abstract: As a general strategy for determining the chemical function of the class of enzymes that cleaves glycosidic linkages with phosphate, the first mass spectrometry and direct detection assay for sugar phosphorylases has been developed and used to study the inhibition and minimal binding requirements of rabbit muscle phosphorylase b. In contrast to the currently employed assays for these enzymes that measure the nonphysiologically relevant reverse reaction of glycosidic bond synthesis and thereby require prior knowledge of not just one but two sugar components, this new method has the potential to greatly reduce the complexity in discovering the substrate specificity of a new enzyme. Certain phosphorylases can catalyze the degradation of glycogen into alpha-D-glucose-1-phosphate and are targets for the development of antidiabetic therapeutics. By electrospray ionization mass spectrometry analysis, the kinetic parameters K(m), V(max), and K(i) (for alpha/beta-D-glucose) have been determined for the rabbit muscle phosphorylase b. This enzyme accepts maltoheptaose, maltohexaose, and maltopentaose as substrates in the direction of glycogen degradation, but the tetrasaccharide maltotetraose cannot serve as a substrate for this phosphorylysis reaction.
authors.affiliation: Department of Chemistry and the Plant Sciences Institute, Iowa State University, Ames, IA 50011, USA.
authors.forename: Corbin J
authors.initials: CJ
authors.lastname: Zea
authors.forename: Nicola L
authors.initials: NL
authors.lastname: Pohl
mesh_terms: D050260:Carbohydrate Metabolism; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D006003:Glycogen / Q000378:metabolism; D007700:Kinetics; D010763:Phosphorylase b / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D006003:Glycogen; D010763:Phosphorylase b; D005947:Glucose