title: Deciphering the kinetic mechanisms controlling selected plant ADP-glucose pyrophosphorylases.
abstract: ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is controlled by thermostability and allosteric regulation. Previous studies suggested that redox affects turnover number and heat stability of AGPases. Here, we investigated how allostery and redox state affect kinetic mechanisms of the reduced, heat labile and the oxidized, heat stable potato tuber enzymes; the heat labile maize endosperm enzyme and a chimeric maize/potato heat stable enzyme that lacks the cysteine responsible for redox changes. With 3-PGA, all AGPases followed a Theorell-Chance Bi Bi mechanism with ATP binding first and ADP-Glc releasing last. 3-PGA increases the binding affinity for both substrates with little effect on velocity for the maize and MP isoforms. By contrast, 3-PGA increases the velocity and the affinity for G-1-P for the potato enzymes. Redox state does not affect kcat of the two potato isoforms. Without 3-PGA the oxidized potato enzyme exhibits a rapid equilibrium random Bi Bi mechanism with a dead end ternary complex. This fundamental change from rapid, ordered binding with little buildup of intermediates to a mechanism featuring relatively slow, random binding is unique to the oxidized potato tuber enzyme. Finally, ADP-Glc the physiologically relevant product of this enzyme has complex, isoform-specific effects on catalysis.
authors.affiliation: Program in Plant Molecular and Cellular Biology and Horticultural Sciences, University of Florida, 1253 Fifield Hall, Gainesville, FL 32611, USA.
authors.forename: Susan K
authors.initials: SK
authors.lastname: Boehlein
authors.forename: Janine R
authors.initials: JR
authors.lastname: Shaw
authors.forename: Seon K
authors.initials: SK
authors.lastname: Hwang
authors.forename: Jon D
authors.initials: JD
authors.lastname: Stewart
authors.forename: L
authors.initials: L
authors.lastname: Curtis Hannah
mesh_terms: D000494:Allosteric Regulation; D056625:Endosperm / Q000201:enzymology; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000737:chemistry; D004795:Enzyme Stability; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000737:chemistry* / Q000235:genetics; D005988:Glyceric Acids / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D010084:Oxidation-Reduction; D010710:Phosphates / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D035281:Plant Tubers / Q000201:enzymology; D021122:Protein Subunits / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D011198:Solanum tuberosum / Q000201:enzymology; D003313:Zea mays / Q000201:enzymology
chemical_list: D020536:Enzyme Activators; D005988:Glyceric Acids; D010710:Phosphates; D010940:Plant Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C005156:3-phosphoglycerate; D051599:Glucose-1-Phosphate Adenylyltransferase