title: A continuous spectrophotometric assay for adenosine 5'-phosphosulfate reductase activity with sulfite-selective probes.
abstract: Mycobacterium tuberculosis (Mtb) adenosine 5'-phosphosulfate (APS) reductase (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of essential reduced sulfur-containing biomolecules, such as cysteine, and is essential for survival in the latent phase of tuberculosis (TB) infection. Despite the importance of APR to Mtb and other bacterial pathogens, current assay methods depend on the use of (35)S-labeled APS or shunt adenosine 5'-monophosphate (AMP) to a coupled-enzyme system. Both methods are cumbersome and require the use of expensive reagents. Here, we report the development of a continuous spectrophotometric method for measuring APR activity by using novel sulfite-selective colorimetric or "off-on" fluorescent levulinate-based probes. Thus, the APR activity can be followed by monitoring the increase in absorbance or fluorescence of the resulting phenolate product. Using this assay, we determined Michaelis-Menten kinetic constants (K(m), k(cat), and k(cat)/K(m)) and the apparent inhibition constant (Ki) for adenosine 5'-diphosphate (ADP), which compared favorably with values obtained in the "gold standard" radioactive assay. The newly developed assay is robust and easy to perform with a simple spectrophotometer.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458, USA.
authors.forename: Hanumantharao
authors.initials: H
authors.lastname: Paritala
authors.forename: Kate S
authors.initials: KS
authors.lastname: Carroll
mesh_terms: D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D007700:Kinetics; D007982:Levulinic Acids / Q000032:analysis*; D016296:Mutagenesis; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000032:analysis*; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods*; D013447:Sulfites / Q000032:analysis*
chemical_list: D005456:Fluorescent Dyes; D007982:Levulinic Acids; D013447:Sulfites; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase; C032246:levulinic acid
keywords: Adenosine 5′-phosphosulfate (APS); Adenosine 5′-phosphosulfate reductase (APR); Levulinate; Sulfate assimilation; Sulfite sensor