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

Abstract

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 k(inact) and K(i), K(i) values are in the micromolar to nanomolar range, and the inactivation half-life is less than 30 s. A Brønsted plot of k(inact)/K(i) has a steep slope (beta(lg) = -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.

Sulfatase-activated fluorophores for rapid discrimination of mycobacterial species and strains

“…We sought further confirmation that the fluorescent bands observed were produced by active sulfatases using sulfatase inhibitors. First, we evaluated a set of arylsulfamates, which are known type I sulfatase inhibitors (48)(49)(50)(51). Inhibition is irreversible and active site-directed, probably through covalent modification of the formylglycine residue (51).…”

“…First, we evaluated a set of arylsulfamates, which are known type I sulfatase inhibitors (48)(49)(50)(51). Inhibition is irreversible and active site-directed, probably through covalent modification of the formylglycine residue (51). Four different arylsulfamates were examined for their ability to inhibit type I sulfatases in mycobacterial lysates.…”

“…5). Arylsulfamates vary in reactivity, with the highest reactivity assigned to compounds with lower pK a values for the parent phenol (51). Coumarin sulfamate (Stx64), a potent inhibitor of human steroid sulfatase (49), was the most reactive compound we examined, with a pK a of 7.8 for the parent phenol.…”

Synthesis of Sulfated Glucosaminides for Profiling Substrate Specificities of Sulfatases and Fungal β‐N‐Acetylhexosaminidases

“…PaAtsA was recombinantly expressed and purified as reported. [43] Potassium p-nitrophenyl sulfate was prepared as reported. [44] Control assays with p-nitrophenyl sulfate: Assays were performed in 1 cm polyacrylate cuvettes and contained potassium p-nitrophenyl sulfate (2 mm) in K 2 HPO 4 /KH 2 PO 4 /0.05 % BSA (50 mm) at pH 6, which also contained a-cyclodextrin (10 mm) to a final volume of 0.5 mL.…”

An Expanded Set of Fluorogenic Sulfatase Activity Probes

“…[8] This limited repertoire stands in contrast to the many fluorogenic probes that have been developed for detection of proteases, [9] phosphatases, [10] esterases, [11] β-lactamases, [3–4, 12] and glycosidases. [13] The hydrolysis product of 4-MUS and DDAO-sulfate, 4-methylumbelliferone (4-MU; λ max = 360, λ emit = 450 nm) and DDAO (λ max = 600, λ emit = 660 nm), respectively, enable fluorescence detection at the extreme ends of the visible spectrum; these products cannot be detected on many low-cost instruments.…”