ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 327 (2004) 252–260 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.01.023 Estimation of binding constants for the substrate and activator of Rhodobacter sphaeroides adenosine 5 0 -diphosphate-glucose pyrophosphorylase using aYnity capillary electrophoresis John Kaddis, a Cecilia Zurita, a Julio Moran, a Margie Borra, b,1 Nephi Polder, b Christopher R. Meyer, b and Frank A. Gomez a,¤ a Department of Chemistry and Biochemistry, California State University at Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8202, USA b Department of Chemistry and Biochemistry, California State University at Fullerton, 800 N. State College Boulevard, Fullerton, CA 92834-6866, USA Received 18 November 2003 Abstract Binding constants were determined for the activator fructose-6-phosphate (F6P) and substrate adenosine 5 0 -triphosphate (ATP) (in the presence and absence of F6P) to the recombinant wild-type (WT) Rhodobacter sphaeroides adenosine 5 0 -diphosphate-(ADP)- glucose pyrophosphorylase (ADPGlc PPase) using aYnity capillary electrophoresis (ACE). In these binding studies, the capillary is initially injected with a plug of sample containing ADPGlc PPase and noninteracting standards. The sample is then subjected to increasing concentrations of F6P or ATP in the running buVer and electrophoresed. Analysis of the change in the migration times of ADPGlc PPase, relative to those of the noninteracting standards, as a function of the varying concentration of F6P or ATP yields a binding constant. The values obtained were in good agreement with kinetic parameters obtained from steady state activity assays. The method was extended to examine the F6P binding constants for the R33A and R22A enzymes and the ATP binding constants for the R8A enzyme in the presence and absence of F6P. The R33A enzyme has been shown by activity assays to be insensitive to F6P activation, indicating a defect in binding or in downstream transmission of the allosteric signal required for full activation. ACE indicated no apparent binding of F6P, supporting the former hypothesis. The R22A enzyme was shown by activity assays to have a »15-fold decrease in apparent aYnity for F6P compared to that of WT while ACE indicated an aYnity comparable to that of WT; potential reasons for this discrepancy are discussed. The R8A enzyme as measured by activity assays exhibits reduced fold-activation by F6P compared to that of WT but increased apparent aYnity for ATP in the presence of F6P. The ACE results were in good agree- ment with the activity assay data, conWrming the increased aYnity for ATP in the presence of F6P. This method demonstrates the quantitative ability of ACE to study diVerent binding sites/ligand interactions in allosteric enzymes.  2004 Elsevier Inc. All rights reserved. Keywords: ADP glucose pyrophosphorylase; AYnity capillary electrophoresis; Binding constants; Dissociation constant (K d ); Relative migration time ratio; Steady state kinetics; Activation ADP-glucose pyrophosphorylase (ADPGlc PPase; 2 EC 2.7.7.27) catalyzes the conversion of glucose-1-phos- phate (Glc-1-P) and ATP to ADPGlc and pyrophos- phate and is the rate-limiting enzyme in the production of biodegradable carbon sources such as glycogen and starch [1,2]. The regulation of bacterial ADPGlc PPase is mediated by the binding and eVect of a number of metabolites which serve as either activators (glycolytic intermediates) or inhibitors (AMP, ADP, P i ) depending on the carbon pathway of the organism. These metabo- lites generally have an eVect on both the V max of the reaction and the apparent aYnities for the substrates [1,2]. ¤ Corresponding author. Fax: 1-323-343-6490. E-mail address: fgomez2@calstatela.edu (F.A. Gomez). 1 Present address: Department of Biomolecular Chemistry, Univer- sity of Wisconsin at Madison, MSC 551, 1300 University Ave., Madi- son, WI 53706-1532, USA. 2 Abbreviations used: ACE, aYnity capillary electrophoresis; ADPGlc PPase, ADP-glucose pyrophosphorylase; RMTR, relative migration time ratio; Glc-1-P, glucose-1-phosphate; F6P, fructose-6-phosphate; EOF, electroosmotic Xow; FBP, fructose-1,6-bisphosphonate.