Direct Evidence for ArO----S Bond Cleavage upon Inactivation of Pseudomonas aeruginosa Arylsulfatase by Aryl Sulfamates

• Published in: Chembiochem : a European journal of chemical biology 2008-03, Vol.9 (4), p.613-623
• Main Authors: Bojarová, Pavla, Denehy, Emma, Walker, Ian, Loft, Karen, De Souza, David P, Woo, L.W. Lawrence, Potter, Barry V.L, McConville, Malcolm J, Williams, Spencer J
• Format: Article
• Language: English
• Subjects: Argon - chemistry; Arylsulfatases - genetics; Arylsulfatases - metabolism; Binding Sites; Enzyme Activation; enzyme catalysis; Hydrogen-Ion Concentration; inhibitors; kinetics; Molecular Structure; Oxygen - chemistry; Phenol - chemistry; Phenol - metabolism; Protein Processing, Post-Translational; Pseudomonas aeruginosa; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; reaction mechanisms; structure-activity relationships; Substrate Specificity; Sulfonic Acids - chemistry; Sulfonic Acids - metabolism; Sulfur - chemistry; Titrimetry
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.200700579

Pseudomonas aeruginosa arylsulfatase catalyses the cleavage of aryl sulfates and is an excellent model for human estrone sulfatase, which is implicated in hormone-dependent breast cancer. Aryl sulfamates are inactivators of sulfatases; however, little is known about their mechanism. We studied the inactivation of Pseudomonas aeruginosa arylsulfatase A by a range of aryl sulfamates, including the clinical agent 667COUMATE (STX64) used to inactivate estrone sulfatase. Inactivation was time dependent, irreversible, and active-site directed, consistent with a covalent modification at the active site. In terms of the kinetic parameters of inactivation kinact and Ki, Ki values are in the micromolar to nanomolar range, and the inactivation half-life is less than 30 s. A Brønsted plot of kinact/Ki has a steep slope (βlg=-1.1), which implies that the transition state for the first irreversible chemical step of inactivation involves a high degree of charge transfer and cleavage of the ArO---S bond. Detection of the released phenol and titration of the residual activity showed the stoichiometry of inactivation to be in the range 3-6, with the greatest values found for the most effective inactivators. Thus, multiple sulfamoylation events appear to occur during the inactivation process. These data provide valuable insight into the mechanism of sulfatase inactivation by sulfamates.