Substrate Inhibition of A. thaliana Adenosine 5′ phosphosulfate kinase by APS occurs via multiple modes

Adenosine‐5′‐phosphosulfate (APS) kinase catalyzes the formation of adenosine 3′phosphate‐5′‐phosphosulfate (PAPS). Steady‐state kinetic studies of APS kinase revealed potent substrate inhibition by APS and suggest that formation of a dead‐end ADP•APS complex causes inhibition. We investigated the energetics of nucleotide binding to APS kinase using isothermal titration calorimetry. APS kinase bound ADP, ATP and AMP‐PNP with similar affinities and enthalpies. APS interaction with E•AMP‐PNP occurs by a sequential binding model to identical sites with a favorable enthalpy. In contrast, the binding isotherm of APS interaction with E•ADP is negatively cooperative and described by a sequential binding model to non‐identical sites. APS binding to the first site was enthalpically unfavorable and enthalpically favorable to the second site. We used stopped‐flow fluorescence to study the kinetics of mantATP and mantADP binding. APS binding to E•mantADP•Pi increased signal, followed by a decrease due to dissociation of mantADP after catalysis. The observed rate of APS binding decreased sigmoidaly with [APS] while the rate of mantADP dissociated decreased hyperbolically. These results suggest that binding to the catalytically active E•mantADP•Pi complex is negatively cooperative, and that substrate inhibition occurs when APS binds to both sites before catalysis.