Experimental and Theoretical Insights into the Mechanisms of Sulfate and Sulfamate Ester Hydrolysis and the End Products of Type I Sulfatase Inactivation by Aryl Sulfamates

Abstract

Type I sulfatases catalyze the hydrolysis of sulfate esters through S-O bond cleavage and possess a catalytically essential formylglycine (FGly) active-site residue that is post-translationally derived from either cysteine or serine. Type I sulfatases are inactivated by aryl sulfamates in a time-dependent, irreversible, and active-site directed manner consistent with covalent modification of the active site. We report a theoretical (SCS-MP2//B3LYP) and experimental study of the uncatalyzed and enzyme-catalyzed hydrolysis of aryl sulfates and sulfamates. In solution, aryl sulfate monoanions undergo hydrolysis by an S(N)2 mechanism whereas aryl sulfamate monoanions follow an S(N)1 pathway with SO2NH as an intermediate; theory traces this difference to the markedly greater stability of SO2NH versus SO3. For Pseudomonas aeruginosa arylsulfatase-catalyzed aryl sulfate hydrolysis, Brønsted analysis (log(V(max)/K(M)) versus leaving group pK(a) value) reveals β(LG) = -0.86 ± 0.23, consistent with an S(N)2 at sulfur reaction but substantially smaller than that reported for uncatalyzed hydrolysis (β(LG) = -1.81). Common to all proposed mechanisms of sulfatase catalysis is a sulfated FGly intermediate. Theory indicates a ≥26 kcal/mol preference for the intermediate to release HSO4(-) by an E2 mechanism, rather than alkaline phosphatase-like S(N)2 substitution by water. An evaluation of the stabilities of various proposed end-products of sulfamate-induced sulfatase inactivation highlights that an imine N-sulfate derived from FGly is the most likely irreversible adduct.

Formylglycine, a Post-Translationally Generated Residue with Unique Catalytic Capabilities and Biotechnology Applications

“…A recent experimental and theoretical study by Krenske and coworkers affirms earlier theoretical studies, 35 which predict a much lower transition state barrier for E2 elimination compared to S N 2 substitution to release sulfate (estimated ΔΔ H ‡ of 26.5 kcal/mol). 36 This study estimates a ΔΔ H ‡ of only 12 kcal/mol for the corresponding phosphoenzyme intermediate, which may account for why fGly is found mostly in sulfatases, and not in alkaline phosphatase. Wild-type ASA is also inhibited by a prohibitively slow rate of elimination/hydrolysis of the corresponding phosphoenzyme intermediate at fGly.…”

“…35 Perhaps inconsistent with this prediction, a measured β LG of 0 for V max but −0.86 for V max / K M for PAS suggests that the alkoxide leaving group is not involved in the rate-determining step but only the first irreversible chemical step. 36 For the desulfation step, hydrolysis of the sulfoenzyme intermediate by water in the S N 2 pathway may be quite slow compared to E2, again for the reason for S-O bond cleavage. Experimentally, pre-steady-state kinetics of the wild-type enzyme may permit the comparison of the relative sulfation/desulfation rates.…”

Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates

“…For both enzymes, the substrate range from PNPS to TS show a difference in V max of five orders of magnitude (Table ). The aryl sulfate esters can be hydrolyzed more readily due to the conjugation afforded by the aromatic ring that provides increased stability for the leaving group . The uncatalyzed hydrolysis of the S–O bond in near‐neutral water for PNPS has been determined as 1.9 × 10 –10 s –1 , and estimated by extrapolation for the n‐pentyl sulfate as 3 × 10 –26 s –1 .…”

“…The aryl sulfate esters can be hydrolyzed more readily due to the conjugation afforded by the aromatic ring that provides increased stability for the leaving group. [37,38] The uncatalyzed hydrolysis of the S-O bond in near-neutral water for PNPS has been determined as 1.9 × 10 -10 s -1 , and estimated by extrapolation for the n-pentyl sulfate as 3 × 10 -26 s -1 . [37] Therefore, even though the maximum rates of hydrolysis for the secondary alkyl sulfate TS is about four to five orders of magnitude slower than the aryl sulfates PNPS and ES, the rate enhancement afforded by HpS or PaS for TS may be significantly greater (~10 11 -fold).…”

Estrogen O-sulfamates and their analogues: Clinical steroid sulfatase inhibitors with broad potential

“…However, the precise mechanism of inhibition is still unknown and other ideas on this subject have been recently discussed [26]. The hydrolysis of EMATE, however, liberates estrone which is estrogenic: the estradiol sulfamate version (E2MATE) behaves similarly with estradiol being liberated which, in rats, resulted in markedly increased systemic (but reduced hepatic) estrogenicity upon oral administration [27] despite the ligand not binding to the estrogen receptor [28].…”